Inhibitors of interleukin 1-beta converting enzyme

ABSTRACT

The present invention relates to novel classes of compounds which are inhibitors of interleukin-1β converting enzyme (“ICE”). This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are particularly well suited for inhibiting ICE activity and consequently, may be advantageously used as agents against interleukin-1-(“IL-1”), apoptosis-, interferon-γ inducing factor-(IGIF), interferon-γ-(“IFN-γ”) mediated diseases, excess dietary alcohol intake diseases, or viral diseases, including inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, and degenerative diseases. This invention also relates to methods for inhibiting ICE activity and decreasing IGIF production and IFN-γ production and methods for treating interleukin-1, apoptosis- and interferon-γ-mediated diseases using the compounds and compositions of this invention. This invention also relates to methods of preparing the compounds of this invention.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to novel classes of compounds whichare inhibitors of interleukin-1β converting enzyme (“ICE”). Thisinvention also relates to pharmaceutical compositions comprising thesecompounds. The compounds and pharmaceutical compositions of thisinvention are particularly well suited for inhibiting ICE activity andconsequently, may be advantageously used as agents againstinterleukin-1-(“IL-1”), apoptosis-, interferon-γ inducing factor-(IGIF),interferon-γ-(“IFN-γ”) mediated diseases, excess dietary alcohol intakediseases, or viral diseases, including inflammatory diseases, autoimmunediseases, destructive bone disorders, proliferative disorders,infectious diseases, and degenerative diseases. This invention alsorelates to methods for inhibiting ICE activity and decreasing IGIFproduction and IFN-γ production and methods for treating interleukin-1,apoptosis- and interferon-γ-mediated diseases using the compounds andcompositions of this invention. This invention also relates to methodsfor preparing the compounds of this invention.

BACKGROUND OF THE INVENTION

[0002] Interleukin 1 (“IL-1”) is a major pro-inflammatory andimmunoregulatory protein that stimulates fibroblast differentiation andproliferation, the production of prostaglandins, collagenase andphospholiphase by synovial cells and chondrocytes, basophil andeosinophil degranulation and neutrophil activation. Oppenheim, J. H. etal., Immunology Today, 7, pp. 45-56 (1986). As such, it is involved inthe pathogenesis of chronic and acute inflammatory and autoimmunediseases. For example, in rheumatoid arthritis, IL-1 is both a mediatorof inflammatory symptoms and of the destruction of the cartilageproteoglycan in afflicted joints. Wood, D. D. et al., Arthritis Rheum.26, 975, (1983); Pettipher, E. J. et al., Proc. Natl. Acad. Sci. U.S.A.71, 295 (1986); Arend, W. P. and Dayer, J. M., Arthritis Rheum. 38, 151(1995). IL-1 is also a highly potent bone resorption agent. Jandiski, J.J., J. Oral Path 17, 145 (1988); Dewhirst, F. E. et al., J. Immunol. 8,2562 1985). It is alternately referred to as “osteoclast activatingfactor” in destructive bone diseases such as osteoarthritis and multiplemyeloma. Bataille, R. et al., Int. J. Clin. Lab. Res. 21(4), 283 (1992).In certain proliferative disorders, such as acute myelogenous leukemiaand multiple myeloma, IL-1 can promote tumor cell growth and adhesion.Bani, M. R., J. Natl. Cancer Inst. 83, 123 (1991); Vidal-Vanaclocha, F.,Cancer Res. 54, 2667 (1994). In these disorders, IL-1 also stimulatesproduction of other cytokines such as IL-6, which can modulate tumordevelopment (Tartour et al., Cancer Res. 54, 6243 (1994). IL-1 ispredominantly produced by peripheral blood monocytes as part of theinflammatory response and exists in two distinct agonist forms, IL-1αand IL-1β. Mosely, B. S. et al., Proc. Nat. Acad. Sci., 84, pp.4572-4576(1987); Lonnemann, G. et al., Eur.J. Immunol., 19, pp.1531-1536 (1989).

[0003] IL-1β is synthesized as a biologically inactive precursor,pIL-1β. pIL-1β lacks a conventional leader sequence and is not processedby a signal peptidase. March, C. J., Nature, 315, pp.641-647 (1985).Instead, pIL-1β is cleaved by interleukin-1β converting enzyme (“ICE”)between Asp-116 and Ala-117 to produce the biologically activeC-terminal fragment found in human serum and synovial fluid. Sleath, P.R., et al., J. Biol. Chem., 265, pp.14526-14528 (1992); A. D. Howard etal., J. Immunol., 147, pp.2964-2969 (1991). ICE is a cysteine proteaselocalized primarily in monocytes. It converts precursor IL-1β to themature form. Black, R. A. et al., FEBS Lett., 247, pp.386-390 (1989);Kostura, M. J. et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.5227-5231(1989). Processing by ICE is also necessary for the transport of matureIL-1β through the cell membrane.

[0004] ICE, or its homologs, also appears to be involved in theregulation of programmed cell death or apoptosis. Yuan, J. et al., Cell,75, pp.641-652 (1993); Miura, M. et al., Cell, 75, pp.653-660 (1993);Nett-Fiordalisi, M. A. et al., J. Cell Biochem., 17B, p.117 (1993). Inparticular, ICE or ICE homologs are thought to be associated with theregulation of apoptosis in neurodegenerative diseases, such asAlzheimer's and Parkinson's disease. Marx, J. and M. Baringa, Science,259, pp.760-762 (1993); Gagliardini, V. et al., Science, 263, pp.826-828(1994). Therapeutic applications for inhibition of apoptosis may includetreatment of Alzheimer's disease, Parkinson's disease, stroke,myocardial infarction, spinal atrophy, and aging.

[0005] ICE has been demonstrated to mediate apoptosis (programmed celldeath) in certain tissue types. Steller, H., Science, 267, p. 1445(1995); Whyte, M. and Evan, G., Nature, 376, p. 17 (1995); Martin, S. J.and Green, D. R., Cell, 82, p. 349 (1995); Alnemri, E. S., et al., J.Biol. Chem., 270, p. 4312 (1995); Yuan, J. Curr. Opin. Cell Biol., 7.,p. 211 (1995). A transgenic mouse with a disruption of the ICE gene isdeficient in Fas-mediated apoptosis (Kuida, K. et al., Science 267, 2000(1995)). This activity of ICE is distinct from its role as theprocessing enzyme for pro-IL1β. It is conceivable that in certain tissuetypes, inhibition of ICE may not affect secretion of mature IL-1β, butmay inhibit apoptosis.

[0006] Enzymatically active ICE has been previously described as aheterodimer composed of two subunits, p20 and p10 (20 kDa and 10 kDamolecular weight, respectively). These subunits are derived from a 45kDa proenzyme (p45) by way of a p30 form, through an activationmechanism that is autocatalytic. Thornberry, N. A. et al., Nature, 356,pp.768-774 (1992). The ICE proenzyme has been divided into severalfunctional domains: a prodomain (p14), a p22/20 subunit, a polypeptidelinker and a plO subunit. Thornberry et al., supra; Casano et al.,Genomics, 20, pp. 474-481 (1994).

[0007] Full length p45 has been characterized by its cDNA and amino acidsequences. PCT patent applications WO 91/15577 and WO 94/00154. The p20and p10 cDNA and amino acid sequences are also known. Thornberry et al.,supra. Murine and rat ICE have also been sequenced and cloned. They havehigh amino acid and nucleic acid sequence homology to human ICE. Miller,D. K. et al., Ann. N.Y. Acad. Sci., 696, pp. 133-148 (1993); Molineaux,S. M. et al., Proc. Nat. Acad. Sci., 90, pp. 1809-1813 (1993). Thethree-dimensional structure of ICE has been determined at atomicresolution by X-ray crystallography. Wilson, K. P., et al., Nature, 370,pp. 270-275 (1994). The active enzyme exists as a tetramer of two p20and two p10 subunits.

[0008] Additionally, there exist human homologs of ICE with sequencesimilarities in the active site regions of the enzymes. Such homologsinclude TX (or ICE_(rel-II) or ICH-2) (Faucheu, et al., EMBO J., 14, p.1914 (1995); Kamens J., et al., J. Biol. Chem., 270, p. 15250 (1995);Nicholson et al., J. Biol. Chem., 270 15870 (1995)), TY (orICE_(rel-III)) (Nicholson et al., J. Biol. Chem., 270, p. 15870 (1995);ICH-1 (or Nedd-2) (Wang, L. et al., Cell, 78, p. 739 (1994)), MCH-2,(Fernandes-Alnemri, T. et al., Cancer Res., 55, p. 2737 (1995), CPP32(or YAMA or apopain) (Fernandes-Alnemri, T. et al., J. Biol. Chem., 269,p. 30761 (1994); Nicholson, D. W. et al., Nature, 376, p. 37 (1995)),and CMH-1 (or MCH-3) (Lippke, et al., J. Biol. Chem., (1996);Fernandes-Alnemri, T. et al., Cancer Res., (1995)). Each of these ICEhomologs, as well as ICE itself, is capable of inducing apoptosis whenoverexpressed in transfected cell lines. Inhibition of one or more ofthese homologs with the peptidyl ICE inhibitorTyr-Val-Ala-Asp-chloromethylketone results in inhibition of apoptosis inprimary cells or cell lines. Lazebnik et al., Nature, 371, p. 346(1994). The compounds described herein are also capable of inhibitingone or more homologs of ICE. Therefore, these compounds may be used toinhibit apoptosis in tissue types that contain ICE homologs.

[0009] Interferon-gamma inducing factor (IGIF) is an approximately18-kDa polypeptide that stimulates T-cell production of interferon-gamma(IFN-γ). IGIF is produced by activated Kupffer cells and macrophages invivo and is exported out of such cells upon endotoxin stimulation. Thus,a compound that decreases IGIF production would be useful as aninhibitor of such T-cell stimulation which in turn would reduce thelevels of IFN-γ production by those cells.

[0010] IFN-γ is a cytokine with immunomodulatory effects on a variety ofimmune cells. In particular, IFN-γ is involved in macrophage activationand Th1 cell selection (F. Belardelli, APMIS, 103, p. 161 (1995)). IFN-γexerts its effects in part by modulating the expression of genes throughthe STAT and IRF pathways (C. Schindler and J. E. Darnell, Ann. Rev.Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer Res. Clin. Oncol.,121, p. 516 (1995)).

[0011] Mice lacking IFN-γ or its receptor have multiple defects inimmune cell function and are resistant to endotoxic shock (S. Huang etal., Science, 259, p.1742 (1993); D.Dalton et al., Science, 259, p.1739(1993); B. D. Car et al., J. Exp. Med., 179, p.1437 (1994)). Along withIL-12, IGIF appears to be a potent inducer of IFN-γ production by Tcells (H. Okamura et al., Infection and Immunity, 63, p.3966 (1995); H.Okamura et al., Nature, 378, p.88 (1995); S. Ushio et al., J.Immunol.,156, p.4274 (1996)).

[0012] IFN-γ has been shown to contribute to the pathology associatedwith a variety of inflammatory, infectious and autoimmune disorders anddiseases. Thus, compounds capable of decreasing IFN-γ production wouldbe useful to ameliorate the effects of IFN-γ related pathologies.

[0013] IGIF is synthesized as a precursor protein, called “pro-IGIF”.Recently, ICE and other members of the ICE/CED-3 family have been linkedto the conversion of pro-IGIF to IGIF or to IFN-γ production in vivo(see PCT patent application WO 97/22619, which is incorporated herein byreference).

[0014] Accordingly, compositions and methods capable of regulating theconversion of pro-IGIF to IGIF would be useful for decreasing IGIF andIFN-γ production in vivo, and thus for ameliorating the detrimentaleffects of these proteins which contribute to human disorders anddiseases.

[0015] ICE inhibitors represent a class of compounds useful for thecontrol of inflammation or apoptosis or both. Peptide and peptidylinhibitors of ICE have been described. PCT patent applications WO91/15577; WO 93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; andEuropean patent application 0 547 699. Such peptidyl inhibitors of ICEhave been observed to block the production of mature IL-1β in a mousemodel of inflammation (vide infra) and to suppress growth of leukemiacells in vitro (Estrov et al., Blood 84, 380a (1994)). However, due totheir peptidic nature, such inhibitors are typically characterized byundesirable pharmacologic properties, such as poor cellular penetrationand cellular activity, poor oral absorption, poor stability and rapidmetabolism. Plattner, J. J. and D. W. Norbeck, in Drug DiscoveryTechnologies, C. R. Clark and W. H. Moos, Eds. (Ellis Horwood,Chichester, England, 1990), pp.92-126. This has hampered theirdevelopment into effective drugs.

[0016] Non-peptidyl compounds have also been reported to inhibit ICE invitro. PCT patent application WO 95/26958; U.S. Pat. Nos. 5,552,400;Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996). However, it isnot clear whether these compounds have the appropriate pharmacologicalprofiles to be therapeutically useful.

[0017] Additionally, current methods for the preparation of suchcompounds are not advantageous. These methods use tributyltin hydride, atoxic, moisture-sensitive reagent. Thus, these methods are inconvenientto carry out, pose a health risk and create toxic-waste disposalproblems. Furthermore, it is difficult to purify compounds prepared bythese methods. A preferred method for preparing compounds, such as theICE inhibitors of this invention, has been described in PCT patentapplication WO 97/22619, which is incorporated herein by reference.

[0018] Accordingly, the need exists for compounds that can effectivelyinhibit the action of ICE in vivo, for use as agents for preventing andtreating chronic and acute forms of IL-1-mediated diseases, apoptosis-,IGIF-, or IFN-γ-mediated diseases, as well as inflammatory, autoimmune,destructive bone, proliferative, infectious, or degenerative diseases.The need also exists for methods of preparing such compounds.

SUMMARY OF THE INVENTION

[0019] The present invention provides novel classes of compounds, andpharmaceutically acceptable derivatives thereof, that are useful asinhibitors of ICE. These compounds can be used alone or in combinationwith other therapeutic or prophylactic agents, such as antibiotics,immunomodulators or other anti-inflammatory agents, for the treatment orprophylaxis of diseases mediated by IL-1, apoptosis, IGIF or IFN-γ.According to a preferred embodiment, the compounds of this invention arecapable of binding to the active site of ICE and inhibiting the activityof that enzyme. Additionally, they have improved cellular potency,improved pharmacokinetics, and/or improved oral bioavailability comparedto peptidyl ICE inhibitors.

[0020] It is a principal object of this invention to provide novelclasses of compounds which are inhibitors of ICE represented by formula:

[0021] wherein the various substituents are described herein.

[0022] It is a further objective of this invention to provide novelprocesses of preparing the compounds of this invention and relatedcompounds.

DETAILED DESCRIPTION OF THE INVENTION

[0023] In order that the invention described herein may be more fullyunderstood, the following detailed description is set forth.

[0024] The following abbreviations and definitions are used throughoutthe application. Abbreviations Ac₂O acetic anhydride n-Bu normal-butylDMF dimethylformamide DIEA N,N-diisopropylethylamine EDC1-(3-Dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride Et₂Odiethyl ether EtOAc ethyl acetate Fmoc 9-fluorenylmethyoxycarbonyl HBTUO-benzotriazol-1-yl-N,N,N,N′- tetramethyluronium hexafluorophosphateHOBT 1-hydroxybenzotriazole hydrate MeOH methanol TFA trifluoroaceticacid

[0025] The terms “HBV”, “HCV” and “HGV” refer to hepatitis-B virus,hepatitis-C virus and hepatitis-G virus, respectively.

[0026] The term “K_(i)” refers to a numerical measure of theeffectiveness of a compound in inhibiting the activity of a targetenzyme such as ICE. Lower values of K_(i) reflect higher effectiveness.The K_(i) value is a derived by fitting experimentally determined ratedata to standard enzyme kinetic equations (see I. H. Segel, EnzymeKinetics, Wiley-Interscience, 1975).

[0027] The term “interferon gamma inducing factor” or “IGIF” refers to afactor which is capable of stimulating the endogenous production ofIFN-γ.

[0028] The term “ICE inhibitor” refers to a compound which is capable ofinhibiting one or more enzymes selected from the group consisting of ICEand ICE homologs. ICE inhibition may be determined using the methodsdescribed and incorporated by reference herein. The skilled practitionerrealizes that an in vivo ICE inhibitor is not necessarily an in vitroICE inhibitor. For example, a prodrug form of a compound typicallydemonstrates little or no activity in in vitro assays. Such prodrugforms may be altered by metabolic or other biochemical processes in thepatient to provide an in vivo ICE inhibitor.

[0029] The term “cytokine” refers to a molecule which mediatesinteractions between cells.

[0030] The term “condition” refers to any disease, disorder or effectthat produces deleterious biological consequences in a subject.

[0031] The term “subject” refers to an animal, or to one or more cellsderived from an animal. Preferably, the animal is a mammal, mostpreferably a human. Cells may be in any form, including but not limitedto cells retained in tissue, cell clusters, immortalized cells,transfected or transformed cells, and cells derived from an animal thathave been physically or phenotypically altered.

[0032] The term “patient” as used in this application refers to anymammal, preferably humans

[0033] The term “alkyl” refers to a straight-chained or branched,saturated aliphatic hydrocarbon containing 1 to 6 carbon atoms.

[0034] The term “alkenyl” refers to a straight-chained or branchedunsaturated hydrocarbon containing 2 to 6 carbons.

[0035] The term “cycloalkyl” refers to a mono- or polycyclic,non-aromatic, hydrocarbon ring system which may optionally containunsaturated bonds in the ring system. Examples include cyclohexyl,adamantyl and norbornyl.

[0036] The term “aryl” refers to a mono- or polycyclic ring system whichcontains 6, 10, 12 or 14 carbons in which at least one ring of the ringsystem is aromatic. The aryl groups of this invention are optionallysingly or multiply substituted with R¹⁷. Examples of aryl ring systemsinclude, phenyl, naphthyl, and tetrahydronaphthyl.

[0037] The term “heteroaryl” refers to a mono- or polycyclic ring systemwhich contains 1 to 15 carbon atoms and 1 to 4 heteroatoms, and in whichat least one ring of the ring system is aromatic. Heteroatoms aresulfur, nitrogen or oxygen. The heteroaryl groups of this invention areoptionally singly or multiply substituted with R¹⁷.

[0038] The term “heterocyclic” refers to a mono- or polycyclic ringsystem which contains 1 to 15 carbon atoms and 1 to 4 heteroatoms, inwhich the mono- or polycyclic ring system may optionally containunsaturated bonds but is not aromatic. Heteroatoms are independentlysulfur, nitrogen, or oxygen.

[0039] The term “alkylaryl” refers to an alkyl group, wherein a hydrogenatom of the alkyl group is replaced by an aryl radical.

[0040] The term “alkylheteroaryl” refers to an alkyl group, wherein ahydrogen atom of the alkyl group is replaced by a heteroaryl radical.

[0041] The term “substitute” refers to the replacement of a hydrogenatom in a compound with a substituent group.

[0042] The term “straight chain” refers to a contiguous unbranchingstring of covalently bound atoms. The straight chain may be substituted,but these substituents are not a part of the straight chain.

[0043] The term “patient” as used in this application refers to anymammal, preferably humans.

[0044] In chemical formulas, parenthesis are used herein to denoteconnectivity in molecules or groups. In particular, parentheses are usedto indicate: 1) that more than one atom or group is bonded to aparticular atom; or 2) a branching point (i.e., the atom immediatelybefore the open parenthesis is bonded both to the atom or group in theparentheses and the atom or group immediately after the closedparenthesis). An example of the first use is “—N(alkyl)₂”, indicatingtwo alkyl groups bond to an N atom. An example of the second use is“—C(O)NH₂”, indicating a carbonyl group and an amino (“NH₂”) group bothbonded to the indicated carbon atom. A “—C(O)NH₂” group may berepresented in other ways, including the following structure:

[0045] Other definitions are set forth in the specification wherenecessary.

COMPOUNDS OF THIS INVENTION

[0046] The compounds of one embodiment (A) of this invention are thoseof formula (I):

[0047] wherein:

[0048] Y is

[0049] provided that if R⁵ is —OH, then Y may also be:

[0050] C is an aryl or a heteroaryl ring, wherein any hydrogen bound toany ring atom is optionally replaced by —R⁴;

[0051] R¹ is -aryl, -heteroaryl, -alkylaryl, or -alkylheteroaryl;

[0052] R² is a bond, —C(O)—, —C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—,—N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—,—N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—, —N(R¹⁹)C(O)C(O)—, or —N(R¹⁹)CH₂C(O)—,provided that if R² is not a bond, then R² is connected to the NHattached to the 7-membered ring through carbonyl or sulfonyl;

[0053] R³ is -aryl, -heteroaryl, -cycloalkyl, -alkyl, —N(alkyl)₂,

[0054] R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H)alkyl, —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O)₂alkyl, or —C(O)alkyl;

[0055] R⁵ is —OH, —OR⁸, or —N(H)OH;

[0056] R⁶ is —H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂N(H)R⁹, —CH₂N(R⁹)R¹², —C(H)N₂,—CH₂F, —CH₂Cl, —C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴;

[0057] R⁸ is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

[0058] R⁹ is —H, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkylheteroaryl, -alkylaryl, -alkylheteroaryl, -aryl, -heteroaryl,or —P(O)R¹⁵R¹⁶;

[0059] R¹⁰ is -alkylaryl, -aryl, -heteroaryl, or -alkylheteroaryl;

[0060] each R¹¹ and R¹² is independently —H, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, or -alkylheteroaryl;

[0061] R¹³ is -alkylaryl, -alkenylaryl, -alkynylaryl, or-alkylheteroaryl;

[0062] R¹⁴ is

[0063] wherein any hydrogen bound to (i) is optionally replaced with R¹⁷and any hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ orR²⁰;

[0064] each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl,-heteroaryl, -cycloalkyl, -alkylaryl, -alkylheteroaryl, -Oalkyl, -Oaryl,-Oheteroaryl, -Oalkylaryl, or -Oalkylheteroaryl;

[0065] R¹⁷ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, —SO₂NH₂, —C(O)H, -alkyl, -cycloalkyl,-perfluoroalkyl, —O-alkyl, —N(H)alkyl, —N(alkyl)₂, —CO₂alkyl,—C(O)N(H)alkyl, —C(O)N(alkyl)₂, —N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl,—N(H)C(O)N(alkyl)₂, —S(O₂)N(H)alkyl, —S(O₂)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, or —C(O)alkyl;

[0066] R¹⁸ is -aryl, -heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl,—O-heteroaryl, —O-alkylaryl, -O-alkylheteroaryl, —N(H)aryl, -N(aryl)₂,—N(H)heteroaryl, —N(Heteroaryl)₂, —N(H)alkylaryl, -N(alkylaryl)₂,—N(H)alkylheteroaryl, -N(alkylheteroaryl)₂, —S-aryl, —S-heteroaryl,-S-alkylaryl, —S-alkylheteroaryl, —C(O)aryl, -C (O)heteroaryl, —C(O)alkylaryl, —C(O) alkyheteroaryl, -CO₂aryl, —CO₂heteroaryl,-Co₂alkylaryl, -CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂,-C(O)N(H)heteroaryl, —C(O)N(heteroaryl)₂, -C(O)N(H)alkylaryl,—C(O)N(alkylaryl)₂, -C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂,—S(O)₂aryl, —S(O)₂heteroaryl, —S(O)₂alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂N(H)aryl, —S(O)₂N(H)heteroaryl, —S(O)₂N(H)alkylaryl,—S(O)₂N(H)alkylheteroaryl, —S(O)2N(aryl)2, —S(O)₂N(heteroaryl)₂,—S(O)₂N(alkylaryl)2, —S(O)₂N(alkylheteroaryl)2, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂,—N(H)C(O)N(alkylheteroaryl)₂;

[0067] R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

[0068] R²⁰ is -alkyl-R¹⁸;

[0069] m is 0 or 1; and

[0070] X is O or S.

[0071] The compounds of another embodiment (B) of this invention arethose of formula (II):

[0072] wherein Y is:

[0073] R⁷ is —C(O)alkyl, —C(O)cycloalkyl, —C(O)alkyenyl, —C(O)alkylaryl,—C(O)alkylheteroaryl, —C(O)heterocycle, or —C(O)alkylheterocycle; andthe other substituents are as described above.

[0074] Preferred compounds of embodiments (A) and (B) are those wherein:

[0075] C is benzo, pyrido, thieno, pyrrolo, furo, imidazo, thiazolo,oxazolo, pyrazolo, isothiazolo, isoxazolo, or triazolo, wherein anyhydrogen bound to any ring atom is optionally replaced by R⁴.

[0076] More preferred compounds of embodiments (A) and (B) are thosewherein:

[0077] y is

[0078] C is benzo, wherein any hydrogen bound to any ring atom isoptionally replaced by R⁴;

[0079] R¹ is -phenyl, -naphthyl, or -isoquinolinyl, wherein R¹⁷ is —OH,—NH₂, —Cl, —F, -Oalkyl, or -N(alkyl)₂;

[0080] R² is —C(O)—, —S(O₂)—, —C(O)C(O)—, or —CH₂C(O)—;

[0081] R³ is -methyl, -ethyl, -n-propyl, -isopropyl, -phenyl,-2-pyridinyl, -3-pyridinyl, -4-pyridinyl, or -thiazolyl;

[0082] R⁴ is -fluoro or -chloro;

[0083] R⁵ is —OH;

[0084] R⁶ is:

[0085] —H; or

[0086] —R¹⁴, wherein X═O, provided that when —R¹⁴ is (i), R¹⁷ is-Oalkyl, —F or —Cl and provided that when —R¹⁴ is (ii), R¹⁸ is -aryl,wherein aryl is phenyl;

[0087] R⁷ is —C(O)alkyl;

[0088] R⁸ is -methyl, -ethyl, -n-propyl, -isopropyl, -cyclopentyl,-phenethyl or -benzyl;

[0089] X is O; or

[0090] m is 0.

[0091] Preferred compounds of embodiment (A) of this invention include,but are not limited to:

[0092] We now prefer compounds of embodiments (C) and (D). The compoundsof embodiment (C) of this invention are those of formula (III):

[0093] wherein:

[0094] Y is

[0095] provided that if R⁵ is —OH, then Y may also be:

[0096] C is an aryl or a heteroaryl ring, wherein any hydrogen bound toany ring atom is optionally replaced by —R⁴;

[0097] R¹ is -aryl, -heteroaryl, -alkylaryl, or -alkylheteroaryl;

[0098] R² is a bond, —C(O)—, —C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—,—N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—,—N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—, —N(R¹⁹)C(O)C(O)—, —N(R¹⁹)CH₂C(O)—, or—C(O)C(═NOR¹¹)—, provided that when R² is not a bond, R² is bonded tothe 7-membered ring NH group through carbonyl or sulfonyl;

[0099] R³ is -aryl, -heteroaryl, -cycloalkyl, -alkyl, —N(alkyl)₂,

[0100] R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H) (alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or CH₂N(alkyl)₂;

[0101] R⁵ is —OH, —OR⁸, or —N(H)OH;

[0102] R⁶ is —H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂NHR⁹, —CH₂N(R⁹)R¹², —CHN₂,—CH₂F, —CH₂Cl, —C(O)N(R¹¹)R¹²—R¹³, or —R¹⁴;

[0103] R⁸ is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

[0104] R⁹ is —H, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkylheteroaryl, -alkylaryl, -alkylheteroaryl, -aryl, -heteroaryl,or —P(O)R¹⁵R¹⁶;

[0105] R¹⁰ is -alkylaryl, -aryl, -heteroaryl, or -alkylheteroaryl;

[0106] each R¹¹ and R¹² is independently —H, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, or -alkylheteroaryl;

[0107] R¹³ is -alkylaryl, -alkenylaryl, -alkynylaryl, or-alkylheteroaryl;

[0108] R¹⁴ is

[0109] wherein any hydrogen bound to (i) is optionally replaced with R¹⁷and any hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ orR²⁰;

[0110] each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl,-heteroaryl, -cycloalkyl, -alkylaryl, -alkylheteroaryl, -Oalkyl, -Oaryl,-Oheteroaryl, -Oalkylaryl, or -Oalkylheteroaryl;

[0111] R¹⁷ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, —S(O₂)NH₂, —C(O)H, -alkyl, -cycloalkyl,-perfluoroalkyl, —O-alkyl, —N(H)alkyl, —N(alkyl)₂, —CO₂alkyl,—C(O)N(H)alkyl, —C(O)N(alkyl)₂, —N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl,—N(H)C(O)N(alkyl)₂, —SO₂N(H)alkyl, —S(O₂)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, or —C(O)alkyl;

[0112] R¹⁸ is -aryl, -heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl,—O-heteroaryl, —O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂,—N(H)heteroaryl, —N(Heteroaryl)₂, —N(H)alkylaryl, —N(alkylaryl)₂,—N(H)alkylheteroaryl, —N(alkylheteroaryl)₂, —S-aryl, —S-heteroaryl,—S-alkylaryl, —S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl,—C(O)alkylaryl, —C(O)alkyheteroaryl, —CO₂aryl, —CO₂heteroaryl,—CO₂alkylaryl, —CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂,—C(O)N(H)heteroaryl, —C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl,—C(O)N(alkylaryl)₂, —C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂,—S(O)₂-aryl, —S(O)₂-heteroaryl, —S(O)₂-alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂NH-aryl, —S(O)₂NH-heteroaryl, —S(O)₂N(H)alkylaryl,—S(O)₂N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O)₂N(H)heteroaryl)₂,—SO₂N(alkylaryl)₂, —SO₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(H)heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂,—N(H)C(O)N(alkylheteroaryl)_(2;)

[0113] R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

[0114] R²⁰ is -alkyl-R¹⁸;

[0115] m is 0 or 1; and

[0116] X is O or S.

[0117] The compound of embodiment (D) of this invention are those offormula (IV):

[0118] wherein Y is:

[0119] R⁷ is —C(O)alkyl, —C(O)cycloalkyl, —C(O)alkyenyl, —C(O)alkylaryl,—C(O)alkylheteroaryl, —C(O)heterocycle, or —C(O)alkylheterocycle; andthe other substituents are as defined above.

[0120] In the above embodiments, R⁸ and R¹⁹ is also independentlyselected from heterocyclyl or alkylcycloalkyl.

[0121] In embodiments (B) and (D) Y is also selected from:

[0122] Preferred compounds of embodiments (C) and (D) are those wherein:

[0123] C is benzo, pyrido, thieno, pyrrolo, furo, imidazo, thiazolo,oxazolo, pyrazolo, isothiazolo, isoxazolo, or triazolo, wherein anyhydrogen bound to any ring atom is optionally replaced by R⁴.

[0124] More preferred compounds of embodiment (C) and (D) are thosewherein:

[0125] Y is

[0126] C is benzo, wherein any hydrogen bound to any ring atom isoptionally replaced by R⁴;

[0127] R¹ is -phenyl, -naphthyl, or -isoquinolinyl, wherein R¹⁷ is —OH,—NH₂, —Cl, —F, -Oalkyl, or —N(alkyl)₂;

[0128] R² is —C(O)—, —S(O)₂—, —C(O)C(O)—, or —CH₂C(O)—;

[0129] R³ is -methyl, -ethyl, -n-propyl, -isopropyl, -phenyl,-2-pyridinyl, -3-pyridinyl, -4-pyridinyl, or -thiazolyl;

[0130] R⁴ is -fluoro or -chloro;

[0131] R⁵ is —OH;

[0132] R⁶ is:

[0133] —H; or

[0134] —R¹⁴, wherein X is O, provided that when —R¹⁴ is (i), R¹⁷ is-Oalkyl, —F or —Cl and provided that when —R¹⁴ is (ii), R¹⁸ is -aryl,wherein aryl is phenyl;

[0135] R⁷ is —C(O)alkyl;

[0136] R⁸ is -methyl, -ethyl, -n-propyl, -isopropyl, -cyclopentyl,-phenethyl or -benzyl;

[0137] X is O; or

[0138] m is 0.

[0139] Other more preferred compounds of embodiments (B) and (D) arethose wherein Y is

[0140] and the other substituents are as defined above.

[0141] Preferred compounds of this invention include, but are notlimited to:

[0142] Another preferred compound of this invention includes, but is notlimited to:

[0143] The ICE inhibitors of this invention may contain one or more“asymmetric” carbon atoms and thus may occur as racemates and racemicmixtures, single enantiomers, diastereomeric mixtures and individualdiastereomers. All such isomeric forms of these compounds are expresslyincluded in the present invention. Each stereogenic carbon may be of theR or S configuration. Although specific compounds and scaffoldsexemplified in this application may be depicted in a particularstereochemical configuration, compounds and scaffolds having either theopposite stereochemistry at any given chiral center or mixtures thereofare also envisioned.

[0144] The ICE inhibitors may optionally be substituted at carbon,nitrogen or other atoms by various substituents. When multiplysubstituted, each substituent may be picked independently of any othersubstituent as long as the combination of substituents results in theformation of a stable compound.

[0145] Combinations of substituents and variables envisioned by thisinvention are only those that result in the formation of stablecompounds. The term “stable”, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and administration toa mammal by methods known in the art. Typically, such compounds arestable at a temperature of 40° C. or less, in the absence of moisture orother chemically reactive conditions, for at least a week.

[0146] Substituents may be represented in various forms. These variousforms are known to the skilled practitioner and may be usedinterchangeably. For example, a methyl substituent on a phenyl ring maybe represented in any of the following forms:

[0147] Various forms of substituents such as methyl are used hereininterchangeably.

[0148] The compounds of this invention have a molecular weight of lessthan or equal to about 700 Daltons, and more preferably between about400 and 600 Daltons. These preferred compounds may be readily absorbedby the bloodstream of patients upon oral administration. This oralavailability makes such compounds excellent agents fororally-administered treatment and prevention regimens against IL-1-,apoptosis-, IGIF-, or IFN-γ-mediated diseases.

[0149] It should be understood that the compounds of this invention mayexist in various equilibrium forms, depending on conditions includingchoice of solvent, pH, and others known to the practitioner skilled inthe art. All such forms of these compounds are expressly included in thepresent invention. In particular, many of the compounds of thisinvention, especially those which contain aldehyde or ketone groups inR₃ and carboxylic acid groups in T, may take hemi-ketal (or hemi-acetal)or hydrated forms. For example, compounds of embodiment (A) take ahemiacetal or hemiketal form when Y is:

[0150] Depending on the choice of solvent and other conditions known tothe practitioner skilled in the art, compounds of this invention mayalso take hydrated, acyloxy ketal, acyloxy acetal, ketal, acetal or enolforms. For example, in embodiment (B) compounds of this invention takehydrated forms when Y is:

[0151] and R⁸ is H;

[0152] acyloxy ketal or acyloxy acetal forms when Y is:

[0153] ketal or acetal forms when Y is:

[0154] and enol forms when Y is:

[0155] In addition, it should be understood that the equilibrium formsof the compounds of this invention may include tautomeric forms. Allsuch forms of these compounds are expressly included in the presentinvention.

[0156] It should be understood that the compounds of this invention maybe modified by appropriate functionalities to enhance selectivebiological properties. Such modifications are known in the art andinclude those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion. In addition, the compounds may be altered to pro-drug formsuch that the desired compound is created in the body of the patient asthe result of the action of metabolic or other biochemical processes onthe pro-drug. Such pro-drug forms typically demonstrate little or noactivity in in vitro assays. Some examples of pro-drug forms includeketal, acetal, oxime, imine and hydrazone forms of compounds whichcontain ketone or aldehyde groups, especially where they occur in the R³group of the compounds of this invention. Other examples of pro-drugforms include the hemi-ketal, hemi-acetal, acyloxy ketal, acyloxyacetal, ketal, acetal and enol forms that are described herein.

[0157] Compositions and Methods

[0158] The compounds of this invention are excellent ligands for ICE.Accordingly, these compounds are capable of targeting and inhibitingevents in IL-1-, apoptosis-, IGIF- and IFN-γ-mediated diseases, and,thus, the ultimate activity of that protein in inflammatory diseases,autoimmune diseases, destructive bone, proliferative disorders,infectious diseases, and degenerative diseases. For example, thecompounds of this invention inhibit the conversion of precursor IL-L1βto mature IL-1β by inhibiting ICE. Because ICE is essential for theproduction of mature IL-1, inhibition of that enzyme effectively blocksinitiation of IL-1-mediated physiological effects and symptoms, such asinflammation, by inhibiting the production of mature IL-1. Thus, byinhibiting IL-1β precursor activity, the compounds of this inventioneffectively function as IL-1 inhibitors.

[0159] Compounds of this invention also inhibit conversion of pro-IGIFinto active, mature IGIF by inhibiting ICE. Because ICE is essential forthe production of mature IGIF, inhibition of ICE effectively blocksinitiation of IGIF-mediated physiological effects and symptoms, byinhibiting production of mature IGIF. IGIF is in turn essential for theproduction of IFN-γ. ICE therefore effectively blocks initiation ofIFN-γ-mediated physiological effects and symptoms, by inhibitingproduction of mature IGIF and thus production of IFN-γ.

[0160] The pharmaceutical compositions and methods of this invention,therefore, will be useful for controlling ICE activity in vivo. Thecompositions and methods of this invention will thus be useful forcontrolling IL-1, IGIF or IFN-γ levels in vivo and for treating orreducing the advancement, severity or effects of IL-1-, apoptosis-,IGIF-, or IFN-γ-mediated conditions, including diseases, disorders oreffects.

[0161] Accordingly, one embodiment of this invention provides a methodfor decreasing IGIF production in a subject comprising the step ofadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of an ICE inhibitor and apharmaceutically acceptable carrier.

[0162] Another embodiment of this invention provides a method fordecreasing IFN-γ production in a subject comprising the step ofadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of an ICE inhibitor and apharmaceutically acceptable carrier.

[0163] In another embodiment, the methods of this invention comprise thestep of administering to a subject a pharmaceutical compositioncomprising an inhibitor of an ICE-related protease that is capable ofcleaving pro-IGIF to active IGIF, and a pharmaceutically acceptablecarrier. One such ICE-related protease is TX, as described above. Thisinvention thus provides methods and pharmaceutical compositions forcontrolling IGIF and IFN-γ levels by administering a TX inhibitor.

[0164] Other ICE-related proteases capable of processing pro-IGIF intoan active IGIF form may also be found. Thus it is envisioned thatinhibitors of those enzymes may be identified by those of skill in theart and will also fall within the scope of this invention.

[0165] Pharmaceutical compositions of this invention comprise an ICEinhibitor or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier, adjuvant or vehicle. Suchcompositions may optionally comprise an additional therapeutic agent.Such agents include, but are not limited to, an anti-inflammatory agent,a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandinor an anti-vascular hyperproliferation compound.

[0166] If the pharmaceutical composition comprises only the ICEinhibitor as the active component, such methods may additionallycomprise the step of administering to the subject an additional agent.Such agents include, but are not limited to, an anti-inflammatory agent,a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandinor an anti-vascular hyperproliferation compound.

[0167] The term “pharmaceutically effective amount” refers to an amounteffective in treating or ameliorating an IL-1-, apoptosis-, IGIF- orIFN-γ-mediated disease in a patient. The term “prophylacticallyeffective amount” refers to an amount effective in preventing orsubstantially lessening IL-1-, apoptosis-, IGIF- or IFN-γ-mediateddiseases in a patient.

[0168] The term “pharmaceutically acceptable carrier or adjuvant” refersto a non-toxic carrier or adjuvant that may be administered to apatient, together with a compound of this invention, and which does notdestroy the pharmacological activity thereof.

[0169] The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ester, or salt of such ester, of acompound of this invention or any other compound which, uponadministration to a recipient, is capable of providing (directly orindirectly) a compound of this invention or an anti-ICE activemetabolite or residue thereof.

[0170] Pharmaceutically acceptable salts of the compounds of thisinvention include, for example, those derived from pharmaceuticallyacceptable inorganic and organic acids and bases. Examples of suitableacids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic,benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids.Other acids, such as oxalic, while not in themselves pharmaceuticallyacceptable, may be employed in the preparation of salts useful asintermediates in obtaining the compounds of the invention and theirpharmaceutically acceptable acid addition salts. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N—(C₁₋₄ alkyl)₄ ⁺ salts.

[0171] This invention also envisions the “quaternization” of any basicnitrogen-containing groups of the compounds disclosed herein. The basicnitrogen can be quaternized with any agents known to those of ordinaryskill in the art including, for example, lower alkyl halides, such asmethyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkylsulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides including benzyl and phenethylbromides. Water or oil-soluble or dispersible products may be obtainedby such quaternization.

[0172] The compounds of this invention may be employed in a conventionalmanner for controlling IGIF and IFN-γ levels in vivo and for treatingdiseases or reducing the advancement or severity of effects which aremediated by IL-1, apoptosis, IGIF or IFN-γ. Such methods of treatment,their dosage levels and requirements may be selected by those ofordinary skill in the art from available methods and techniques.

[0173] For example, a compound of this invention may be combined with apharmaceutically acceptable adjuvant for administration to a patientsuffering from an IL-1-, apoptosis-, IGIF- or IFN-γ-mediated disease ina pharmaceutically acceptable manner and in an amount effective tolessen the severity of that disease.

[0174] Alternatively, the compounds of this invention may be used incompositions and methods for treating or protecting individuals againstIL-1, apoptosis-, IGIF, or IFN-γ mediated diseases over extended periodsof time. The compounds may be employed in such compositions either aloneor together with other compounds of this invention in a mannerconsistent with the conventional utilization of ICE inhibitors inpharmaceutical compositions. For example, a compound of this inventionmay be combined with pharmaceutically acceptable adjuvantsconventionally employed in vaccines and administered in prophylacticallyeffective amounts to protect individuals over an extended period of timeagainst IL-1-, apoptosis-, IGIF, or IFN-γ mediated diseases.

[0175] The compounds of this invention may also be co-administered withother ICE inhibitors to increase the effect of therapy or prophylaxisagainst various IL-1-, apoptosis-, IGIF- or IFN-γ mediated diseases.

[0176] In addition, the compounds of this invention may be used incombination either conventional anti-inflammatory agents or with matrixmetalloprotease inhibitors, lipoxygenase inhibitors and antagonists ofcytokines other than IL-1β.

[0177] The compounds of this invention can also be administered incombination with immunomodulators (e.g., bropirimine, anti-humanalpha-interferon antibody, IL-2, GM-CSF, methionine enkephalin,interferon-alpha, diethyldithiocarbamate, tumor necrosis factor,naltrexone and EPO), with prostaglandins, or with antiviral agents(e.g., 3TC, polysulfated polysaccharides, ganiclovir, ribavirin,acyclovir, alpha interferon, trimethotrexate and fancyclovir) orprodrugs of these or related compounds to prevent or combatIL-1-mediated disease symptoms such as inflammation.

[0178] When the compounds of this invention are administered incombination therapies with other agents, they may be administeredsequentially or concurrently to the patient. Alternatively,pharmaceutical or prophylactic compositions according to this inventioncomprise a combination of an ICE inhibitor of this invention and anothertherapeutic or prophylactic agent.

[0179] Pharmaceutically acceptable carriers, adjuvants and vehicles thatmay be used in the pharmaceutical compositions of this inventioninclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat andself-emulsifying drug delivery systems (SEDDS) such as α-tocopherol,polyethyleneglycol 1000 succinate, or other similar polymeric deliverymatrices.

[0180] The pharmaceutical compositions of this invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir. Weprefer oral administration. The pharmaceutical compositions of thisinvention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compoundor its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injection or infusiontechniques.

[0181] The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as those described in Pharmacopeia Helvetica, Ph.Helv, or a similar alcohol.

[0182] The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

[0183] The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but-are not limited to, cocoa butter, beeswax and polyethyleneglycols.

[0184] Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-administered transdermalpatches are also included in this invention.

[0185] The pharmaceutical compositions of this invention may beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

[0186] Dosage levels of between about 0.01 and about 100 mg/kg bodyweight per day, preferably between 0.5 and about 75 mg/kg body weightper day and most preferably between about 1 and 50 mg/kg body weight perday of the active ingredient compound are useful in a monotherapy forthe prevention and treatment of IL-1-, apoptosis-, IGIF- and IFN-γmediated diseases, including inflammatory diseases, autoimmune diseases,destructive bone disorders, proliferative disorders, infectiousdiseases, degenerative diseases, necrotic diseases, osteoarthritis,acute pancreatitis, chronic pancreatitis, asthma, adult respiratorydistress syndrome, glomerulonephritis, rheumatoid arthritis, systemiclupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease,autoimmune gastritis, insulin-dependent diabetes mellitus (Type I),autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia,chronic active hepatitis, myasthenia gravis, inflammatory bowel disease,Crohn's disease, psoriasis, graft vs. host disease, osteoporosis,multiple myeloma-related bone disorder, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer'sdisease, Parkinson's disease, cerebral ischemia, myocardial ischemia,spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis,HIV-related encephalitis, aging, alopecia, neurological damage due tostroke, ulcerative collitis, infectious hepatitis, juvenile diabetes,lichenplanus, acute dermatomyositis, eczema, primary cirrhosis, uveitis,Behcet's disease, atopic skin disease, pure red cell aplasia, aplasticanemia, amyotrophic lateral sclerosis, nephrotic syndrome and systemicdiseases or diseases with effects localized in the liver or other organshaving an inflammatory or apoptotic component caused by excess dietaryalcohol intake or viruses, such as HBV, HCV, HGV, yellow fever virus,dengue fever virus, and Japanese encephalitis virus.

[0187] Typically, the pharmaceutical compositions of this invention willbe administered from about 1 to 5 times per day or alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

[0188] When the compositions of this invention comprise a combination ofan ICE inhibitor and one or more additional therapeutic or prophylacticagents, both the ICE inhibitor and the additional agent should bepresent at dosage levels of between about 10% to 80% of the dosagenormally administered in a monotherapy regime.

[0189] Upon improvement of a patient's condition, a maintenance dose ofa compound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence or disease symptoms.

[0190] As the skilled artisan will appreciate, lower or higher dosesthan those recited above may be required. Specific dosage and treatmentregimens for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health status, sex, diet, time ofadministration, rate of excretion, drug combination, the severity andcourse of the disease, and the patient's disposition to the disease andthe judgment of the treating physician.

[0191] The IL-1 mediated diseases which may be treated or prevented bythe compounds of this invention include, but are not limited to,inflammatory diseases, autoimmune diseases, proliferative disorders,infectious diseases, and degenerative diseases. The apoptosis-mediateddiseases which may be treated or prevented by the compounds of thisinvention include degenerative diseases.

[0192] IL-1 mediated inflammatory diseases which may be treated orprevented include, but are not limited to osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, and adult respiratorydistress syndrome. Preferrably the inflammatory disease isosteoarthritis or acute pancreatitis.

[0193] IL-1 mediated autoimmune diseases which may be treated orprevented include, but are not limited to, glomeralonephritis,rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Graves' disease, autoimmune gastritis, insulin-dependentdiabetes mellitus (Type I), autoimmune hemolytic anemia, autoimmuneneutropenia, thrbmbocytopenia, chronic active hepatitis, myastheniagravis, multiple sclerosis, inflammatory bowel disease,,Crohn's disease,psoriasis, and graft vs. host disease. Preferrably the autoimmunedisease is rheumatoid arthritis, inflammatory bowel disease, Crohn'sdisease, or psoriasis,

[0194] IL-1 mediated destructive bone disorders which may be treated orprevented include, but are not limited to, osteoporosis and multiplemyeloma-related bone disorder.

[0195] IL-1 mediated proliferative diseases which may be -treated orprevented include, but are not limited to, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, andmultiple myeloma.

[0196] IL-1 mediated infectious diseases which may be treated orprevented include, but are not limited to, sepsis, septic shock, andShigellosis.

[0197] The IL-1-mediated degenerative or necrotic diseases which may betreated or prevented by the compounds of this invention include, but arenot limited to, Alzheimer's disease, Parkinson's disease, cerebralischemia, and myocardial ischemia. Preferably, the degenerative diseaseis Alzheimer's disease.

[0198] The apoptosis-mediated degenerative diseases which may be treatedor prevented by the compounds of this invention include, but are notlimited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia,myocardial ischemia, spinal muscular atrophy, multiple sclerosis,AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia,and neurological damage due to stroke.

[0199] Other diseases having an inflammatory or apoptotic component maybe treated or prevented by the compounds of this invention. Suchdiseases may be systemic diseases or diseases with effects localized inthe liver or other organs and may be caused by, for example, excessdietary alcohol intake or viruses, such as HBV, HCV, HGV, yellow fevervirus, dengue fever virus, and Japanese encephalitis virus.

[0200] The IGIF- or IFN-γ-mediated diseases which may be treated orprevented by the compounds of this invention include, but are notlimited to, inflammatory, infectious, autoimmune, proliferative,neurodegenerative and necrotic conditions.

[0201] IGIF- or IFN-γ-mediated inflammatory diseases which may betreated or prevented include, but are not limited to osteoarthritis,acute pancreatitis, chronic pancreatitis, asthma, rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, ulcerative collitis,cerebral ischemia, myocardial ischemia and adult respiratory distresssyndrome. Preferrably, the inflammatory disease is rheumatoid arthritis,ulcerative collitis, Crohn's disease, hepatitis or adult respiratorydistress syndrome.

[0202] IGIF- or IFN-γ-mediated infectious diseases which may be treatedor prevented include, but are not limited to infectious hepatitis,sepsis, septic shock and shigellosis.

[0203] IGIF- or IFN-γ-mediated autoimmune diseases which may be treatedor prevented include, but are not limited to glomerulonephritis,systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves'disease, autoimmune gastritis, insulin-dependent diabetes mellitus (TypeI), juvenile diabetes, autoimmune hemolytic anemia, autoiimmuneneutropenia, thrombocytopenia, myasthenia gravis, multiple sclerosis,psoriasis, lichenplanus, graft vs. host disease, acute dermatomyositis,eczema, primary cirrhosis, hepatitis, uveitis, Behcet's disease, atopicskin disease, pure red cell aplasia, aplastic anemia, amyotrophiclateral sclerosis and nephrotic syndrome. Preferrably, the autoimmunedisease is glomerulonephritis, insulin-dependent diabetes mellitus (TypeI), juvenile diabetes, psoriasis, graft vs. host disease or hepatitis.

[0204] Although this invention focuses on the use of the compoundsdisclosed herein for preventing and treating IL-1, apoptosis-, IGIF,IFN-γ-mediated diseases, the compounds of this invention can also beused as inhibitory agents for other cysteine proteases.

[0205] The compounds of this invention are also useful as commercialreagents which effectively bind to ICE or other cysteine proteases. Ascommercial reagents, the compounds of this invention, and theirderivatives, may be used to block proteolysis of a target peptide inbiochemical or cellular assays for ICE and ICE homologs or may bederivatized to bind to a stable resin as a tethered substrate foraffinity chromatography applications. These and other uses whichcharacterize commercial cysteine protease inhibitors will be evident tothose of ordinary skill in the art.

[0206] Process of Preparing N-Acylamino Compounds

[0207] The ICE inhibitors of this invention may be synthesized usingconventional techniques. Advantageously, these compounds areconveniently synthesized from readily available starting materials.

[0208] The compounds of this invention are among the most readilysynthesized ICE inhibitors known. Many of the previously described ICEinhibitors contain four or more chiral centers and numerous peptidelinkages. The relative ease with which the compounds of this inventioncan be synthesized represents an advantage in the large scale productionof these compounds.

[0209] For example, compounds of this invention may be prepared usingthe processes described herein. As can be appreciated by the skilledpractitioner, these processes are not the only means by which thecompounds described and claimed in this application may be synthesized.Further methods will be evident to those of ordinary skill in the art.Additionally, the various synthetic steps described herein may beperformed in an alternate sequence or order to give the desiredcompounds.

[0210] A preferred method for preparing the N-acylamino compounds ofthis invention comprise the steps of:

[0211] a) mixing a carboxylic acid with an N-alloc-protected amine inthe presence of an inert solvent, triphenylphoshine, a nucleophilicscavenger, and tetrakis-triphenyl phosphine palladium(0) at ambienttemperature under an inert atmosphere; and

[0212] b) adding to the step a) mixture, HOBT and EDC; and optionallycomprising the further step of:

[0213] c) hydrolyzing the step b) mixture in the presence of a solutioncomprising an acid and H₂O, wherein the step b) mixture is optionallyconcentrated, prior to hydrolyzing.

[0214] Preferably, the inert solvent is CH₂Cl₂, DMF, or a mixture ofCH₂Cl₂ and DMF.

[0215] Preferably, the nucleophilic scavenger is dimedone, morpholine,trimethylsilyl dimethylamine, or dimethyl barbituric acid. Morepreferably, the nucleophilic scavenger is trimethylsilyl dimethylamineor dimethyl barbituric acid.

[0216] Preferably, the solution comprises trifluoroacetic acid in about1-90% by weight. More preferably, the solution comprises trifluoroaceticacid in about 20-50% by weight.

[0217] Alternatively, the solution comprises hydrochloric acid in about0.1-30% by weight. More preferably, the solution comprises hydrochloricacid in about 5-15% by weight.

[0218] More preferably, in the above process, the inert solvent isCH₂Cl₂, DMF, or a mixture of CH₂Cl₂ and DMF and the nucleophilicscavenger is dimedone, morpholine, trimethylsilyl dimethylamine, ordimethyl barbituric acid.

[0219] Most preferably, in the above process the inert solvent isCH₂Cl₂, DMF, or a mixture of CH₂Cl₂ and DMF and the nucleophilicscavenger is trimethylsilyl dimethylamine or dimethyl barbituric acid.

[0220] In an example of a preferred process, the N-acylamino compound isrepresented by formula (V):

[0221] wherein:

[0222] R²¹ is

[0223] R²² is:

[0224] m is 1; and

[0225] R²³ is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or alkylheterocycle and the other substituents are asdescribed above.

[0226] Preferably, the carboxylic acid is R²²—OH and the N-allocprotected amine is:

[0227] (IV):

[0228] wherein R²³ and m are as defined above.

[0229] In order that this invention be more fully understood, thefollowing examples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLE 1

[0230]

[0231](3S)-3-[3(R,S)-1,3-Dihydro-2-oxo-5-phenyl((benzyloxycarbonyl)amino)-2H-1,4-benzodiazepin-1-acetylamino]-4-oxobutyricAcid

[0232] Step 1. A 0° C. THF solution (30 ml) of 1 (2.4 g 6.22 mmol;prepared as described in Sherrill and Sugg, J. Org. Chem., 60, pp. 730-4(1995)) was treated with NaH (240 mg, 6.00 mmol of a 60% oildispersion). After the reaction was stirred for 1 hr at 0° C., methylbromoacetate (0.6 ml, 6.32 mmol) was added to the reaction and theallowed to warm to room temperature. The reaction was quenched withwater (10 ml) and aqueous 10% NaHSO₄ (1 ml) and extracted with ethylacetate (2×). The combined organic layers were dried over anhydrousNa₂SO₄ and concentrated in vacuo. Chromatography (SiO₂, 10 to 3%methylene chloride/ethyl acetate eluent) gave 2.15 g (76%) of 2.

[0233] Step 2. Aqueous 1 N NaOH (3.5 ml, 3.5 mmol) was added to asolution of 2 (340 mg, 0.74 mmol) in methanol and THF (6 ml of 1:1). Thereaction was stirred at room temperature for 18 hr. The reaction wasevaporated, dissolved in water and acidified with aqueous 10% NaHSO₄ topH 3. The aqueous layer was extracted with ethyl acetate (3×) and thecombined organic layers were dried anhydrous Na₂SO₄ and concentrated invacuo to give 210 mg (64%) of 3.

[0234] Step 3. (3S) 3-(1-Fluorenylmethoxycarbonylamino)-4-oxobutyricacid tert-butyl ester semicarbazone (4; 226 mg, 0.5 mmol; prepared in asimilar manner as the benzyloxycarbonly analog described in Graybilletal. Int. J. Protein Res., 44, pp. 173-82 (1994)) was dissolved in 10ml of acetonitrile (20 ml) and diethylamine (2 ml) was added to thesolution. The reaction was stirred for two hours, concentrated in vacuo,the resulting dissolved in acetonitrile and concentrated in vacuo againto give (3S) 3-amino-4-oxobutyric acid tert-butyl ester semicarbazone. A5° C. solution of the semicarbazone and 3 (188 mg, 0.424 mmol) inmethylene chloride/DMF (6 ml of 1:1) was treated with1-hydroxybenzotriazole (HOBt; 57 mg, 0.424 mmol) and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC; 115mg, 0.6 mmol) and the reaction was stirred at room temperature for 16hr. The reaction was diluted with ethyl acetate (100 ml) and washed withwater, aqueous saturated NaHCO₃ and aqueous saturated NaCl, dried overdried over anhydrous Na₂SO₄ and concentrated in vacuo. Chromatography(SiO2, 5% ammonium hydroxide/5% methanol/methylene chloride eluent) gave250 mg of 5

Example 1

[0235] Step 4. Semicarbazone 5 (250 mg) was dissolved in 25%TFA/methylene chloride (10 ml) and stirred at room temperature for 5 hr.to give a yellow foam that was dissolved in 5 ml MeOH, 1 ml acetic Acid,and 1 ml of 37% aqueous formaldehyde that was stirred at roomtemperature for 18 hrs. The reaction was concentrated in vacuo and theresulting gum purified by chromatography (SiO₂, 1% formic acid/2%methanol/methylene chloride eluent) to afford 69 mg (30%) of Example 1from compound 3. ¹H-NMR (CD3OD) δ 2.42-2.54 (m, 1H); 2.60-2.76 (m, 1H);4.22-4.38 (m, 1H); 4.39-4.48 (m, 0.5H); 4.5-4.75 (m, 2.5H); 5.15 (s,2H); 5.32 (br. s, 1H); 7.2-7.86 (m, 14H).

Example 2

[0236] Further data for Example 2 is found in Table 1.

[0237](3S)-3-[3(R,S)-1,3-Dihydro-2-oxo-5-phenyl-((3,5-dichloro-4-methoxybenzoyl)amino)-2H-1,4-benzodiazepin-1-acetylamino]-4-oxobutyricAcid

[0238] Step 1. MBHA resin (0.63 mmol/g, 4.14 g, 2.61 mmol) was suspendedin dimethylacetamide (20 mL) followed by addition of 6 (2.37 g, 4.0mmol, prepared from (3S) 3-(fluorenylmethyloxycarbonyl) -4-oxobutryicacid t-butyl ester according to A.M. Murphy et. al. J. Am. Chem. Soc.,114, 3156-3157 (1992)), O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU; 1.53 g, 4.04 mmol), and DIEA (1.75 mL, 10.0mmol). The reaction mixture was agitated 3 hr at room temperature usinga wrist arm shaker. The resin was isolated on a sintered glass funnel bysuction filtration and washed with dimethylacetamide (6×20 mL).Unreacted amine groups were then capped by reacting the resin with 20%(v/v) acetic anhydride/dimethylformamide (2×25 ml) directly in thefunnel (10 min wash). The resin was washed with dimethylformamide (3×50ml), dichloromethane (3×50 ml) and methanol (3×20 mL) prior to dryingovernight in vacuo to yield 7 (5.33 g, 0.35 mmol/g, 1.86 mmol, 71%).

[0239] Step 2. Resin 7 (4.0 g, 0.35 mmol/g, 1.4 mmol) was swelled in asintered glass funnel by washing with dimethylformamide (3×25 mL). TheFmoc protecting group was then cleaved with 25% (v/v)piperidine/dimethylformamide (25 mL) for 10 min (intermittent stirring)and then for 20 min with fresh piperidine reagent (25 ml). The resin wasthen washed with dimethylformamide (3×25 ml), followed byN-methypyrrolidone (2×25 mL). After transferring the resin to a 100 mLflask, N-methypyrrolidone was added to obtain a slurry followed by 8(0.958 g, 2.1 mmol), HOBT·H2O (0.321 g, 2.1 mmol), HBTU (0.796 g, 2.1mmol) and DIEA (0.732 mL, 4.2 mmol). The reaction mixture was agitatedovernight at room temperature using a wrist arm shaker. The resinwork-up was performed as described for 7 to yield 9 (4.17 g, 0.27mmol/g, 1.12 mmol, 80%).

[0240] Step 3. This compound was prepared from resin 9 (0.17 g, 0.047mmol) using an Advanced ChemTech 396 Multiple Peptide synthesizer. Theautomated cycles consisted of a resin wash with dimethylformamide (3×1mL), deprotection with 25% (v/v) piperidine in dimethylformamide (1 mL)for 3 min followed by fresh reagent (1 mL) for 10 min. The resin 10 waswashed with dimethylformamide (3×1 mL) and N-methypyrrolidone (3×1 mL).

[0241] Step 4. Resin 10 was acylated with a solution of 0.4M3,5-dichloro-4-methoxybenzoic acid and 0.4M HOBT in N-methypyrrolidone(0.5 mL), a solution of 0.4M HBTU in N-methylpyrrolidone (0.5 mL) and asolution of 1.6M DIEA in N-methypyrrolidone (0.25 mL) and the reactionwas shaken for 2 hr at room temperature. The acylation step wasrepeated. Finally, the resin was washed with dimethylformamide (3×1 mL),dichloromethane (3×1 mL) and dried in vacuo yield resin 11.

[0242] Step 5. The aldehyde was cleaved from the resin 11 and globallydeprotected by treatment with 95% TFA/5% H₂O (v/v, 1.5 mL) for 30 min atroom temperature. After washing the resin with cleavage reagent (1 mL),the combined filtrates were concentrated to dryness in a Savant AES2000SpeedVac. The resulting pellets were dissolved in 50% acetonitrile/50%H₂O/0.1% TFA (5 mL) and lyophilized to obtain the crude product as anoff-white solid. The compound was purified by semi-prep RP-HPLC with aWaters DeltaPak C8 300A column (15 μ, 30×300 mm) eluting with a linearacetonitrile gradient (20%-70%) containing 0.1% TFA (v/v) over 45 min at22 mL/min. Fractions containing the desired product were pooled andlyophilized to provide Example 2 (14.1 mg, 23.1 μmol, 49%).

Example 3

[0243] Further data for Example 3 is found in Table 1.

[0244](3S)-3-[3(R,S)-1,3-Dihydro-2-oxo-5-phenyl(benzoylamino)-2H-1,4-benzodiazepin-1-acetylamino]-4-oxobutyricAcid

[0245] Step 4. Following a similar procedure as method 1, resin 5 wasacylated with 0.5M benzoyl chloride in N-methypyrrolidone (1 mL) and1.6M DIEA in N-methypyrrolidone (0.35 mL) for 3 hr at room temperature.The acylation step was repeated to yield resin 12. This same methodologywas also applied to the preparation of sulfonylamino compounds byreplacing benzoyl chloride with sulfonyl chlorides. This samemethodology was also applied to the preparation of urea compounds byreplacing benzoyl chloride with the appropriate isocyanate.

[0246] Step 5. Cleavage of the aldehyde from the resin 12 and workupgave Example 3 (31.6 mg).

Examples 4-27

[0247] Examples 4-27 were prepared by methods similar to the methodsused to prepare Examples 2 or 3 (see Table 1). TABLE 1 HPLC RT (min)Prep. Ex. Structure MF MW Purity (%) MS Example 2

C29H24Cl2N4O7 611.44 13.8 88% (M + H) +617.2 2 3

C28H24N4O6 512.53 10.43 96% (M + H) +513.9 3 4

C30H27N5O7 569.58  8.93 98% (M + H) +570.1 2 5

C30H26ClN5O7 604.02  9.96 98% (M + H) +605.4 2 6

C31H30N4O7 570.61 12.25 94% (M + H) +572.3 2 7

C28H23Cl2N5O6 596.43 11.9 98% (M + H) +597.8 2 8

C29H26N4O7 542.55 10.6 94% (M + H) +543.9 2 9

C30H29N5O6 555.60  9.35 93% (M + H) +556.5 2 10

C29H26N4O7 542.55 10.78 94% (M + H) +544.2 2 11

C31H25N5O6 563.57 11.8 96% (M + H) +565.1 2 12

C29H24N4O8 556.54 10.66 97% (M + H) +558.1 2 13

C29H26N4O6 526.55 10.36 94% (M + H) +528.1 3 14

C28H30N4O6 518.57 10.75 94% (M +Na) +541.9 3 15

C30H28N4O6 540.58 11.22 98% (M + H) +541.6 3 16

C28H25N5O6 527.54 10.24 97% (M + H) +529.0 3 17

C29H27N5O6 541.57  9.60 98% (M + H) +542.7 3 18

C28H24ClN5O6 561.99 10.50 95% (M + H) +562.7 2 19

C30H28N4O7 556.58 10.42 98% (M + H) +557.5 2 20

C28H24N4O7 528.53  9.00 98% (M + H) +529.4 2 21

C2 H22Cl2N4O7 597.42 11.55 98% (M + H) +598.7 2 23

C35H24Cl4N4O9 786.41 16.07 97% (M + H) +787.2 2 24

C27H24N4O7S 548.58 11.02 98% (M + H) +549.9 3 25

C29H24N4O7 540.54 11.63 98% (M + H) +542.0 2 26

C32H26N4O6 562.59 12.23 98% (M + H) +563.8 2 27

C32H26N4O6 562.59 12.88 98% (M + H) +564.1 3

Example 28 ICE Inhibition

[0248] We obtained inhibition constants (K_(i)) and IC₅₀ for compoundsof this invention using the three described below (Examples 28 and 31).Table 2 this data for Examples 1-20 and 21-27. TABLE 2 UV-Visible CellPBMC Whole human blood Example Ki (nM) IC50 (nM) IC50 (nM) 1 650 20000 2250 3 1500 4 525 5 300 6 180 7 30 >20000 8 500 9 150 10 160 11 140 12600 13 1000 14 7500 15 4000 16 4000 17 4000 18 55 >20000 19 20 10000 20155 21 2.5 10000 23 90 24 300 25 250 26 100 27 45

[0249] 1. Enzyme Assay with UV-Visible Substrate

[0250] This assay is run using anSuccinyl-Tyr-Val-Ala-Asp-p-Nitroanilide substrate. Synthesis ofanalogous substrates is described by L. A. Reiter (Int. J. PeptideProtein Res., 43, pp. 87-96 (1994)). The assay mixture contains:

[0251] 65 μl buffer (10 mM Tris, 1 mM DTT, 0.1% CHAPS @pH 8.1)

[0252] 10 μl ICE (50 nM final concentration to give a rate of ˜1mOD/min)

[0253] 5 μl DMSO/Inhibitor mixture

[0254] 20 μl 400 μM Substrate (80 μM final concentration)

[0255] 100 μl total reaction volume

[0256] The visible ICE assay is run in a 96-well microtiter plate.Buffer, ICE and DMSO (if inhibitor is present) are added to the wells inthe order listed. The components are left to incubate at roomtemperature for 15 minutes starting at the time that all components arepresent in all wells. The microtiter plate reader is set to incubate at37° C. After the 15 minute incubation, substrate is added directly tothe wells and the reaction is monitored by following the release of thechromophore (pNA) at 405-603 nm at 37° C. for 20 minutes. A linear fitof the data is performed and the rate is calculated in mOD/min. DMSO isonly present during experiments involving inhibitors, buffer is used tomake up the volume to 100 μl in the other experiments.

[0257] 2. Enzyme Assay with Fluorescent Substrate

[0258] This assay is run essentially according to Thornberry et al.Nature 356 pp. 768-774 (1992), using substrate 17 referenced in thatarticle. The substrate is: Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin(AMC). The following components are mixed:

[0259] 65 μl buffer (10 mM Tris, 1 mM DTT, 0.1% CHAPS @pH 8.1)

[0260] 10 μl ICE (2-10 nM final concentration)

[0261] 5 μl DMSO/inhibitor solution

[0262] 20 μl 150 μM Substrate (30 μM final)

[0263] 100 μl total reaction volume

[0264] The assay is run in a 96-well microtiter plate. Buffer and ICEare added to the wells. The components are left to incubate at 37° C.for 15 minutes in a temperature-controlled wellplate. After the 15minute incubation, the reaction is started by adding substrate directlyto the wells and the reaction is monitored at 37° C. for 30 minutes byfollowing the release of the AMC fluorophore using an excitationwavelength for 380 nm and an emission wavelength of 460 nm. A linear fitof the data for each well is performed and a rate is determined influorescence units per second.

[0265] For determination of enzyme inhibition constants (K_(i)) or themode of inhibition (competitive, uncompetitive or noncompetitive), therate data determined in the enzyme assays at varying inhibitorconcentrations are computer-fit to standard enzyme kinetic equations(see I. H. Segel, Enzyme Kinetics, Wiley-Interscience, 1975).

[0266] The determination of second order rate constants for irreversibleinhibitors was performed by fitting the fluorescence vs time data to theprogress equations of Morrison. Morrison, J. F., Mol Cell. Biophys., 2,pp. 347-368 (1985). Thornberry et al. published a description of thesemethods for measurement of rate constants of irreversible inhibitors ofICE. Thornberry, N. A., et al. Biochemistry, 33, pp. 3923-3940 (1994).For compounds where no prior complex formation can be observedkinetically, the second order rate constants (k_(inact)) are deriveddirectly from the slope of the linear plots of k_(obs) vs. inhibitorconcentration [I]. For compounds where prior complex formation to theenzyme can be detected, the hyperbolic plots of k_(obs) vs. [I] are fitto the equation for saturation kinetics to first generate K_(i) and k′.The second order rate constant k_(inact) is then given by k′/K_(i).

[0267] 3. PBMC Cell Assay

[0268] IL-1β Assay with a Mixed Population of Human Peripheral BloodMononuclear Cells (PBMC) or Enriched Adherent Mononuclear Cells

[0269] Processing of pre-IL-1β by ICE can be measured in cell cultureusing a variety of cell sources. Human PBMC obtained from healthy donorsprovides a mixed population of lymphocyte subtypes and mononuclear cellsthat produce a spectrum of interleukins and cytokines in response tomany classes of physiological stimulators. Adherent mononuclear cellsfrom PBMC provides an enriched source of normal monocytes for selectivestudies of cytokine production by activated cells.

[0270] Experimental Procedure:

[0271] An initial dilution series of test compound in DMSO or ethanol isprepared, with a subsequent dilution into RPMI-10% FBS media (containing2 mM L-glutamine, 10 mM HEPES, 50 U and 50 ug/ml pen/strep) respectivelyto yield drugs at 4× the final test concentration containing 0.4% DMSOor 0.4% ethanol. The final concentration of DMSO is 0.1% for all drugdilutions. A concentration titration which brackets the apparent K_(i)for a test compound determined in an ICE inhibition assay is generallyused for the primary compound screen.

[0272] Generally 5-6 compound dilutions are tested and the cellularcomponent of the assay is performed in duplicate, with duplicate ELISAdeterminations on each cell culture supernatant.

[0273] PBMC Isolation and IL-1 Assay:

[0274] Buffy coat cells isolated from one pint human blood (yielding40-45 ml final volume plasma plus cells) are diluted with media to 80 mland LeukoPREP separation tubes (Becton Dickinson) are each overlaid with10 ml of cell suspension. After 15 min centrifugation at 1500-1800×g,the plasma/media layer is aspirated and then the mononuclear cell layeris collected with a Pasteur pipette and transferred to a 15 ml conicalcentrifuge tube (Corning). Media is added to bring the volume to 15 ml,gently mix the cells by inversion and centrifuge at 300×g for 15 min.The PBMC pellet is resuspended in a small volume of media, the cells arecounted and adjusted to 6×106 cells/ml.

[0275] For the cellular assay, 1.0 ml of the cell suspension is added toeach well of a 24-well flat bottom tissue culture plate (Corning), 0.5ml test compound dilution and 0.5 ml LPS solution (Sigma #L-3012; 20ng/ml solution prepared in complete RPMI media; final LPS concentration5 ng/ml). The 0.5 ml additions of test compound and LPS are usuallysufficient to mix the contents of the wells. Three control mixtures arerun per experiment, with either LPS alone, solvent vehicle control,and/or additional media to adjust the final culture volume to 2.0 ml.The cell cultures are incubated for 16-18 hr at 37° C. in the presenceof 5% CO₂.

[0276] At the end of the incubation period, cells are harvested andtransferred to 15 ml conical centrifuge tubes. After centrifugation for10 min at 200×g, supernatants are harvested and transferred to 1.5 mlEppendorf tubes. It may be noted that the cell pellet may be utilizedfor a biochemical evaluation of pre-IL-10 and/or mature IL-1β content incytosol extracts by Western blotting or ELISA with pre-IL-10 specificantisera.

[0277] Isolation of Adherent Mononuclear Cells:

[0278] PBMC are isolated and prepared as described above. Media (1.0 ml)is first added to wells followed by 0.5 ml of the PBMC suspension. Aftera one hour incubation, plates are gently shaken and nonadherent cellsaspirated from each well. Wells are then gently washed three times with1.0 ml of media and final resuspended in 1.0 ml media. The enrichmentfor adherent cells generally yields 2.5-3.0×10⁵ cells per well. Theaddition of test compounds, LPS, cell incubation conditions andprocessing of supernatants proceeds as described above.

[0279] Elisa:

[0280] We have used Quantikine kits (R&D Systems) for measurement ofmature IL-1β. Assays are performed according to the manufacturer'sdirections. Mature IL-1β levels of about 1-3 ng/ml in both PBMC andadherent mononuclear cell positive controls are observed. ELISA assaysare performed on 1:5, 1:10 and 1:20 dilutions of supernatants fromLPS-positive controls to select the optimal dilution for supernatants inthe test panel.

[0281] The inhibitory potency of the compounds can be represented by anIC₅₀ value, which is the concentration of inhibitor at which 50% ofmature IL-1β is detected in the supernatant as compared to the positivecontrols.

[0282] The skilled practitioner realizes that values obtained in cellassays, such as those described herein, can depend on multiple factors.The values may not necessarily represent fine quantitative results.

Example 29 Pharmacokinetic Studies in the Mouse

[0283] Peptidyl ICE inhibitors are cleared rapidly with clearance ratesgreater than 100 μ/min/kg. Compounds with lower clearance rates haveimproved pharmacokinetic properties relative to peptidyl ICE inhibitors.

[0284] Clearance rates for compounds of this invention (μ/min/kg) may beobtained using the method described below:

[0285] Sample Preparation and Dosing

[0286] Compounds are dissolved in sterile TRIS solution (0.02M or 0.05M)at a concentration of 2.5 mg/ml. Where necessary to ensure a completesolution, the sample is first dissolved in a minimum ofdimethylacetamide (maximum of 5% of total solution volume) then dilutedwith the TRIS solution.

[0287] The drug solution is administered to CD-1 mice (Charles RiverLaboratories -26-31 g) via the tail vein at a dose volume of 10 ml/kggiving a drug dose of 25 mg/kg, for example.

[0288] Mice may be dosed in groups (of 5, for example) for eachtimepoint (generally from 2 minutes to 2 hours) and then at theappropriate time the animals are anaesthetised with halothane and theblood collected into individual heparinized tubes by jugular severance.The blood samples are cooled to 0° C. then the plasma separated andstored at −20° C. until assayed.

[0289] Bioassay

[0290] Drug concentration in the plasma samples are determined by HPLCanalysis with UV or MS (ESP) detection. Reverse phase chromatography isemployed using a variety of bonded phases from C1 to C18 with eluentscomposed of aqueous buffer/acetonitrile mixtures run under isocraticconditions.

[0291] Quantitation is by external standard methods with calibrationcurves constructed by spiking plasma with drug solutions to giveconcentrations in the range of 0.5 to 50 μg/ml.

[0292] Prior to analysis the plasma samples are deproteinated by theaddition of acetonitrile, methanol, trichloroacetic acid or perchloricacid followed by centrifugation at 10,000 g for 10 minutes. Samplevolumes of 20 μl to 50 μl are injected for analysis.

[0293] Representative Dosinq and Sampling Procedure

[0294] The drug is dissolved in sterile 0.02M Tris to give a 2.5 mg/mlsolution which is administered to 11 groups of 5 male CD-1 mice via thetail vein at a dose of 25 mg/kg. At each of the following timepoints: 2,5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes a group of animals isanaesthetised and the blood collected into heparinized tubes. Afterseparation the plasma is stored at −20° C. until assayed.

[0295] Representative Assay

[0296] Aliquots of plasma (150 μl) are treated with 5% perchloric acid(5 μl) then mixed by vortexing and allowing to stand for 90 minutesprior to centrifugation. The resulting supernatant is separated and 20μl is injected for HPLC analysis.

[0297] Representative HPLC Conditions Column 100 × 4.6 mm Kromasil KR100 5C4 Mobile Phase 0.1 m Tris pH7.5 86% Acetonitrile 14% Flowrate 1ml/min Detection UV at 210 nm Retention Time 3.4 mins

Example 30

[0298] Peptidyl ICE inhibitors are cleared rapidly with clearance ratesgreater than 80 ml/min/kg. Compounds with lower clearance rates haveimproved pharmacokinetic properties relative to peptidyl ICE inhibitors.

[0299] The rate of clearance in the rat (ml/min/kg) for compounds ofthis invention may be obtained using the method described below:

In Vivo Rat Clearance Assay

[0300] Representative Procedure

[0301] Cannulations of the jugular and carotid vessels of rats underanesthesia are performed one day prior to the pharmacokinetic study. M.J. Free, R. A. Jaffee; ‘Cannulation techniques for the collection bloodand other bodily fluids’; in: Animal Models; p. 480-495; N. J.Alexander, Ed.; Academic Press; (1978). Drug (10 mg/mL) is administeredvia the jugular vein in a vehicle usually consisting of: propyleneglycol/saline, containing 100 mM sodium bicarbonate in a 1:1 ratio.Animals are dosed with 10-20 mg drug/kg and blood samples are drawn at0, 2, 5, 7, 10, 15, 20, 30, 60, and 90 minutes from an indwellingcarotid catheter. The blood is centrifuged to plasma and stored at −20°C. until analysis. Pharmacokinetic analysis of data is performed bynon-linear regression using standard software such as RStrip (MicroMathSoftware, UT) and/or Pcnonlin (SCI Software, NC) to obtain clearancevalues.

[0302] Representative Analytical:

[0303] Rat plasma is extracted with an equal volume of acetonitrile(containing 0.1% TFA). Samples are then centrifuged at approximately1,000×g and the supernatant analyzed by gradient HPLC. A typical assayprocedure is described below.

[0304] 200 μL of plasma is precipitated with 200 μL of 0.1%trifluoroacetic acid (TFA) in acetonitrile and 10 μL of a 50% aqueouszinc chloride solution, vortexed then centrifuged at −1000×g and thesupernatant collected and analyzed by HPLC. HPLC procedure: Column:Zorbax SB-CN (4.6 × 150 mm) (5μ particle size) Column temperature: 50°C. Flow rate: 1.0 mL/min Injection volume: 75 μL. Mobile phase: A = 0.1%TFA in water and B = 100% acetonitrile Gradient employed: 100% A to 30%A in 15.5 min  0% A at 16 min 100% A at 19.2 min Wavelength: 214 nm

[0305] A standard curve is run at 20, 10, 5, 2 and 1 μg/mLconcentrations.

Example 31 Whole Blood Assay for IL-1β Production

[0306] Whole blood assay ICSO values for compounds of this invention areobtained using the method described below:

[0307] Purpose:

[0308] The whole blood assay is a simple method for measuring theproduction of IL-1β (or other cytokines) and the activity of potentialinhibitors. The complexity of this assay system, with its fullcomplement of lymphoid and inflammatory cell types, spectrum of plasmaproteins and red blood cells is an ideal in vitro representation ofhuman in vivo physiologic conditions.

[0309] Materials:

[0310] Pyrogen-free syringes (˜30 cc)

[0311] Pyrogen-free sterile vacuum tubes containing lyophilized

[0312] Na₂EDTA (4.5 mg/10 ml tube)

[0313] Human whole blood sample (˜30-50 cc)

[0314] 1.5 ml eppendorf tubes

[0315] Test compound stock solutions (˜25 mM in DMSO or other solvent)

[0316] Endotoxin -free sodium chloride solution (0.9%) and HBSS

[0317] Lipopolysaccharide (Sigma; Cat.# L-3012) stock solution at 1mg/ml in HBSS

[0318] IL-1β ELISA Kit (R & D Systems; Cat # DLB50)

[0319] TNFα (ELISA Kit (R & D Systems; Cat # DTA50)

[0320] Water bath or incubator

[0321] Whole Blood Assay Experimental Procedure:

[0322] Set incubator or water bath at 30° C. Aliquot 0.25 ml of bloodinto 1.5 ml eppendorf tubes, making sure to invert the whole bloodsample tubes after every two aliquots. Differences in replicates mayresult if the cells sediment and are not uniformly suspended. Use of apositive displacement pipette will also minimize differences betweenreplicate aliquots.

[0323] Prepare drug dilutions in sterile pyrogen-free saline by serialdilution. A dilution series which brackets the apparent K_(i) for a testcompound determined in an ICE inhibition assay is generally used for theprimary compound screen. For extremely hydrophobic compounds, preparecompound dilutions in fresh plasma obtained from the same blood donor orin PBS-containing 5% DMSO to enhance solubility.

[0324] Add 25 μl test compound dilution or vehicle control and gentlymix the sample. Then add 5.0 μl LPS solution (250 ng/ml stocked preparedfresh: 5.0 ng/ml final concentration LPS), and mix again. Incubate thetubes at 30° C. in a water bath for 16-18 hr with occasional mixing.Alternatively, the tubes can be placed in a rotator set at 4 rpm for thesame incubation period. This assay should be set up in duplicate ortriplicate with the following controls: negative control—no LPS;positive control—no test inhibitor; vehicle control—the highestconcentration of DMSO or compound solvent used in the experiment.Additional saline is added to all control tubes to normalize volumes forboth control and experimental whole blood test samples.

[0325] After the incubation period, whole blood samples are centrifugedfor 10 minutes at ˜2000 rpm in the microfuge, plasma is transferred to afresh microfuge tube and centrifuged at 1000×g to pellet residualplatelets if necessary. Plasma samples may be stored frozen at −70° C.prior to assay for cytokine levels by ELISA.

[0326] ELISA:

[0327] R & D Systems (614 McKinley Place N.E. Minneapolis, Minn. 55413)Quantikine kits may be used for measurement of IL-1β and TNF-α. Theassays are performed according to the manufacturer's directions. IL-1βlevels of ˜1-5 ng/ml in positive controls among a range of individualsmay be observed. A 1:200 dilution of plasma for all samples is usuallysufficient for experiments for ELISA results to fall on the linear rangeof the ELISA standard curves. It may be necessary to optimize standarddilutions if you observe differences in the whole blood assay. Nerad, J.L. et al., J. Leukocyte Biol., 52, pp. 687-692 (1992).

Example 32 Inhibition of ICE Homologs

[0328] 1. Isolation of ICE Homoloqs

[0329] Expression of TX in Insect Cells Using a Baculovirus ExpressionSystem.

[0330] Tx cDNA (C. Faucheu et al., EMBO, 14, p. 1914 (1995)) issubcloned into a modified pVL1393 transfer vector, co-transfected theresultant plasmid (pVL1393/TX) into insect cells with viral DNA and therecombinant baculovirus is identified. After the generation of hightiter recombinant virus stock, the medium is examined for TX activityusing the visible ICE assay. Typically, infection of Spodopterafrugiperda (Sf9) insect cells at an MOI of 5 with recombinant virusstock result in a maximum expression after 48 hours of 4.7 μg/ml. ICE isused as a standard in the assay.

[0331] Amino terminal T7 tagged versions of ICE or TX are alsoexpressed. Designed originally to assist the identification andpurification of the recombinant proteins, the various constructs alsoallow examination of different levels of expression and of the relativelevels of apoptosis experienced by the different homologs. Apoptosis inthe infected Sf9 cells (examined using a Trypan Blue exclusion assay) isincreased in the lines expressing ICE or TX relative to cells infectedwith the viral DNA alone.

[0332] Expression and Purification of N-Terminally (His)₆-tagged CPP32in E. coli.

[0333] A cDNA encoding a CPP32 (Fernanes-Alnemri et al., supra, 1994)polypeptide starting at Ser (29) is PCR amplified with primers that addin frame XhoI sites to both the 5′ and 3′ ends of the cDNA and theresulting XhoI fragment ligated into a Xho I-cut pET-15b expressionvector to create an in frame fusion with (his)₆ tag at the N-terminus ofthe fusion protein. The predicted recombinant protein starts with theamino acid sequence of MGSSHHHHHHSSG{umlaut over (LVPRGS)}HMLE, whereLVPRGS represents a thrombin cleavage site, followed by CPP32 startingat Ser (29). E. coli BL21(DE3) carrying the plasmid are grown to logphase at 30° C. and are then induced with 0.8 mM IPTG. Cells areharvested two hours after IPTG addition. Lysates are prepared andsoluble proteins are purified by Ni-agarose chromatography. All of theexpressed CPP32 protein would be in the processed form. N-terminalsequencing analysis should indicate that the processing has occurred atthe authentic site between Asp (175) and Ser (176). Approximately 50 μgof CPP32 protein from 200 ml culture could be obtained. As determined byactive site titration, the purified proteins are fully active. Theprotease preparations are also very active in vitro in cleaving PARP aswell as the synthetic DEVD-AMC substrate (Nicholson et al., 1995).

[0334] 2. Inhibition. of ICE Homologs

[0335] The selectivity of a panel of reversible inhibitors for ICEhomologs may be obtained. ICE enzyme assays.are performed according toWilson et al. (1994) using a YVAD-AMC substrate (Thornberry et al.,1992). Assay of TX activity is performed using the ICE substrate underidentical conditions to ICE. Assay of CPP32 is performed using aDEVD-AMC substrate (Nicholson et al., 1995).

[0336] Second-order rate constants for inactivation of ICE and ICEhomologs with irreversible inhibitors are obtained.

Example 33 Inhibition of apoptosis

[0337] Fas-Induced Apoptosis in U937 Cells.

[0338] Compounds may be evaluated for their ability to blockanti-Fas-induced apopotosis. Using RT-PCR, mRNA encoding ICE, TX, ICH-1,CPP32 and CMH-1 in unstimulated U937 cells may be detected. This cellline may be used for apoptosis studies. For example, U937 cells areseeded in culture at 1×10⁵ cells/ml and grown to −5×10⁶ cells/ml. Forapoptosis experiments, 2×10⁶ cells are plated in 24-well tissue cultureplates in 1 ml RPMI-1640-10% FBS and stimulated with 100 ng/ml anti-Fasantigen antibody (Medical and Biological Laboratories, Ltd.). After a 24hr incubation at 37° C., the percentage of apoptotic cells is determinedby FACS analysis using ApoTag reagents.

[0339] All compounds are tested initially at 20 μM and titrations areperformed with active compounds to determine IC₅₀ values.

Example 34

[0340] In Vivo Acute Assay for Efficacy as Anti-Inflammatory Agent

[0341] LPS-Induced IL-1β Production.

[0342] Efficacy is evaluated in CD1 mice (n=6 per condition, forexample) challenged with LPS (20 mg/kg IP). The test compounds areprepared in olive oil:DMSO:ethanol (90:5:5) and administered by IPinjection one hour after LPS. Blood is collected seven hours after LPSchallenge. Serum IL-1β levels are measured by ELISA.

[0343] Compounds may also be administered by oral gavage to assessabsorption. Compounds administered orally that inhibit IL-1β secretionare suggestive of the potential oral efficacy of those compounds as ICEinhibitors and thus as anti-inflammatory agents.

Example 35 Measurement of Blood Levels of Prodrugs

[0344] Mice are administered a p.o. (oral) dose of compounds (50 mg/kg,for example) prepared in 0.5% carboxymethylcellulose. Blood samples arecollected at 1 and 7 hours after dosing. Serum is extracted byprecipitation with an equal volume of acetonitrile containing 2% formicacid followed by centrifugation. The supernatant is analyzed by liquidchromatography-mass spectrometry (ESI-MS) with a detection level of 0.03to 3 μg/ml. Detectable blood levels are thus determined.

Example 36 ICE Inhibition Assays—IGIF

[0345] IGIF may be substituted for IL-1 in the ICE inhibition assaysdescribed in Example 28. Thus, the ability of ICE inhibitors to decreaseIGIF production may be determined.

[0346] For example, to run the human PBMC assay, human buffy coat cellsmay be obtained from blood donors and peripheral blood mononuclear cells(PBMC) isolated by centrifugation in LeukoPrep tubes (Becton-Dickinson,Lincoln Park, N.J.). PBMC are added (3×10⁶/well) to 24 well Corningtissue culture plates and after 1 hr incubation at 37° C., non-adherentcells are removed by gently washing. Adherent mononuclear cells arestimulated with LPS (1 μg/ml) with or without ICE inhibitor in 2 mlRPMI-1640-10% FBS. After 16-18 hr incubation at 37° C., IGIF and IFN-γare quantitated in culture supernatants by ELISA.

Example 37

[0347] The antiviral efficacy of compounds may be evaluated in variousin vitro and in vivo assays. For example, compounds may be tested in invitro viral replication assays. In vitro assays may employ whole cellsor isolated cellular components. In vivo assays include animal modelsfor viral diseases. Examples of such animal models include, but are notlimited to, rodent models for HBV or HCV infection, the Woodchuck modelfor HBV infection, and chimpanzee model for HCV infection.

[0348] ICE inhibitors may also be evaluated in animal models for dietaryalcohol-induced disease.

Examples 38-59

[0349] Examples 38-56 (Table 3) were prepared by methods similar to themethods used to prepare Examples 2 or 3. Examples 57-59 (Table 3) wereprepared by methods similar to the methods used to prepare Example 1.TABLE 3 Ex. Structure 38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

ICE Inhibition

[0350] We obtained inhibition constants (K_(i)) and IC₅₀ values forcompounds of this invention using the methods described herein (seeExamples 28 and 31). Table 4 lists this data for Examples 11, 38-56 and58-59. TABLE 4 UV-Visible Cell PBMC Whole human blood Example Ki (nM)IC50 (nM) IC50 (nM) 11 140 9000 38 590 39 260 >20000 40 100 41 350 421200 43 1700 44 2400 45 450 46 180 47 1500 48 2300 49 48 >8000 5040 >8000 51 1000 52 1500 53 700 54 300 55 7500 56 135 58 48 >20000 59 609200

Example 61 Mouse Carrageenan Peritoneal Inflammation RepresentativeProcedure

[0351] Inflammation is induced in mice with an intraperitoneal (IP)injection of 10 mg carrageenan in 0.58 mL of saline (Griswold et al.,Inflammation, 13, pp. 727-739 (1989)). Drugs are administered by oralgavage in ethanol/PEG/water, β-cyclodextrin, labrosol/water orcremophor/water vehicle. The mice are sacrificed at 4 hours postcarrageenan administration, then injected IP with 2 ml of salinecontaining 5 U/ml heparin. After gentle massage of the peritoneum, asmall incision is made, the contents collected and volume recorded.Samples are kept on ice until centrifuged (130×g, 8 mins at 4° C.) toremove cellular material, and the resultant supernatant stored at −20°C. IL-1β levels in the peritoneal fluid are determined by ELISA.

Example 62 Type II Collagen-Induced Arthritis

[0352] Representative Procedure

[0353] Type II collagen-induced arthritis is established in male DBA/1Jmice at described Wooley and Geiger (Wooley, P. H., Methods inEnzymology, 162, pp. 361-373 (1988) and Geiger, T., Clinical andExperimental Rheumatology, 11, pp. 515-522 (1993)). Chick sternum TypeII collagen (4 mg/kg in 10 mM acetic acid) is emulsified with an equalvolume of Freund's complete adjuvant (FCA) by repeated passages (400)between two 10 ml glass syringes with a gauge 16 double-hub needle. Miceare immunized by intradermal injection (501; 1001 CII per mouse) ofcollagen emulsion 21 days later at the contra-lateral side of the tailbase. Drugs are administered twice a day (10, 25 and 50 mg/kg) by oralgavage approximately 7 h apart. Vehicles that may be used includeethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/water.Drug treatments are initiated within 2 h of the CII boosterimmunization. Inflammation is scored on a 1 to 4 scale of increasingseverity on the two front paws and the scores are added to give thefinal score.

Example 63 In vivo Bioavailability Determination

[0354] Representative Procedure

[0355] The drugs (10-100 mg/kg) are dosed orally to rats (10 mL/kg) inethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/water.Blood samples are drawn from the carotid artery at 0.25, 0.50, 1, 1.5,2, 3, 4, 6, and 8 hours after dosing, centrifuged to plasma and storedat −70° C. until analysis. Aldehyde concentrations are determined usingan enzymatic assay. Pharmacokinetic analysis of data is performed bynon-linear regression using RStrip (MicroMath Software, UT). Drugavailability values are determined as follows: (AUC of drug after oralprodrug dosing/AUC of drug after i.v. dosing of drug)x(dose i.v./dosep.o.)×100%.

[0356] The data of the examples above demonstrate that compoundsaccording to this invention display inhibitory activity towards IL-1βConverting Enzyme and that ICE controls IGIF and IFN-γ levels.

[0357] Insofar as the compounds of this invention are able to inhibitICE in vitro and furthermore, may be delivered orally to mammals, theyare of evident clinical utility for the treatment of IL-1-, apoptosis-,IGIF-, and IFN-γ-mediated diseases. These tests are predictive of thecompounds ability to inhibit ICE in vivo.

[0358] While we have described a number of embodiments of thisinvention, it is apparent that our basic constructions may be altered toprovide other embodiments which utilize the products and processes ofthis invention.

What is claimed is:
 1. A compound represented by formula (III):

wherein: Y is

provided that if R⁵ is —OH, then Y may also be:

C is an aryl or a heteroaryl ring, wherein any hydrogen bound to anyring atom is optionally replaced by —R⁴; R¹ is -aryl, -heteroaryl,-alkylaryl, or -alkylheteroaryl; R² is a bond, —C(O)—, —C(O)C(O)—,—S(O)₂—, —OC(O)—, —N(H)C(O)—, —N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—,—OCH₂C(O)—, —N(H)CH₂C(O)—, —N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—,—N(R¹⁹)C(O)C(O)—, —N(R¹⁹)CH₂C(O)—, or —C(O)C(═NOR¹¹)—, provided thatwhen R² is not a bond, R² is bonded to the 7-membered ring NH groupthrough carbonyl or sulfonyl; R³ is -aryl, -heteroaryl, -cycloalkyl,-alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H) (alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or CH₂N(alkyl)₂; R⁵ is—OH, —OR⁸, or —N(H)OH; R⁶ is —H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂NHR⁹,—CH₂N(R⁹)R¹², —(H)N₂, —CH₂F, —CH₂Cl, —C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴; R⁸is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or alkylheterocycle; R⁹ is —H, —C(O)aryl,—C(O)heteroaryl, —C (O)alkylaryl, —C(O) alkylheteroaryl, -alkylaryl,-alkylheteroaryl, -aryl, -heteroaryl, or —P(O)R¹⁵R¹⁶; R¹⁰ is -alkylaryl,-aryl, -heteroaryl, or -alkylheteroaryl; each R¹¹ and R¹² isindependently —H, -alkyl, -aryl, -heteroaryl, -cycloalkyl, -alkylaryl,or -alkylheteroaryl; R¹³ is -alkylaryl, -alkenylaryl, -alkynylaryl, or-alkylheteroaryl; R¹⁴ is

wherein any hydrogen bound to (i) is optionally replaced with R¹⁷ andany hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ or R²⁰;each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, -alkylheteroaryl, -Oalkyl, -Oaryl,-Oheteroaryl, -Oalkylaryl, or -Oalkylheteroaryl; R¹⁷ is —OH, —F, —Cl,—Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂, —N(H)C(O)H, —N(H)C(O)NH₂,—S(O)₂NH₂, —C(O)H, -alkyl, -cycloalkyl, -perfluoroalkyl, —O-alkyl,—N(H)alkyl, —N(alkyl)₂, —CO₂alkyl, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S(O)₂N(H)alkyl,—S(O)₂N(alkyl)2, —S-alkyl, —S(O₂)alkyl, or —C(O)alkyl; R¹⁸ is -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl, —O-heteroaryl,—O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂, —N(H)heteroaryl, —N(Heteroaryl) 2′—N(H) alkylaryl, —N(alkylaryl)₂, —N(H)alkylheteroaryl, —N (alkylheteroaryl)₂, —S-aryl, —S-heteroaryl,—S-alkylaryl, —S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl, —C(O) alkyheteroaryl, —CO₂aryl, —CO₂heteroaryl,—CO₂alkylaryl, —CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂,—C(O)N(H)heteroaryl, —C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl,—C(O)N(alkylaryl)₂, —C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂,—S(O)₂-aryl, —S(O)₂-heteroaryl, —S(O)₂-alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂N(H) aryl, —S(O₂)N(H)heteroaryl, —S(O₂)N(H)alkylaryl,—SO)₂N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O)₂N(H) (heteroaryl)₂,—S(O)₂N(alkylaryl)₂, —S(O)₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂,—N(H)C(O)N(alkylheteroaryl)₂; R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, or -alkylheterocycle; R²⁰ is-alkyl-R¹⁸; m is 0 or 1; and X is O or S.
 2. A compound represented byformula (IV),

wherein Y is:

C is an aryl or a heteroaryl ring, wherein any hydrogen bound to anyring atom is optionally replaced by —R⁴; R¹ is -aryl, -heteroaryl,-alkylaryl, or -alkylheteroaryl; R² is a bond, —C(O)—, —C(O)C(O)—,—S(O)₂—, —OC(O)—, —N(H)C(O)—, —N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—,—OCH₂C(O)—, —N(H)CH₂C(O)—, -N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—,—N(R¹⁹)C(O)C(O)—, —N(R¹⁹)CH₂C(O)—, or —C(O)C(=NOR¹¹)—, provided thatwhen R² is not a bond, R² is bonded to the 7-membered ring NH groupthrough carbonyl or sulfonyl; R³ is -aryl, -heteroaryl, -cycloalkyl,-alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H)(alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or CH₂N(alkyl)₂; R⁶ is—H, —CH₂OR⁹, —CH₂SR¹⁰—CH₂N(H)R⁹, —CH₂N(R⁹)R¹², —CHN₂, —CH₂F, —CH₂Cl,—C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴; R⁷ is —C(O)alkyl, —C(O)cycloalkyl,—C(O)alkyenyl, —C(O)alkylaryl, —C(O)alkylheteroaryl, —C(O)heterocycle,or —C(O)alkylheterocycle; R⁸ is -alkyl, -cycloalkyl, -aryl, -heteroaryl,-alkylaryl, -alkylheteroaryl, or alkylheterocycle; R⁹ is —H, —C(O)aryl,—C(O)heteroaryl, —C(O)alkylaryl, —C(O)alkylheteroaryl, -alkylaryl,-alkylheteroaryl, or —P(O)R¹⁵R¹⁶; R¹⁰ is -alkylaryl or -alkylheteroaryl;each R¹¹ and R¹² is independently —H, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, or -alkylheteroaryl; R¹³ is -alkylaryl,-alkenylaryl, -alkynylaryl, or -alkylheteroaryl; R¹⁴ is

wherein any hydrogen bound to (i) is optionally replaced with R¹⁷ andany hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ or R²⁰;each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, -alkylheteroalkyl, -Oalkyl, -Oaryl,-Oheteroaryl, -Oalkylaryl, or -Oalkylheteroaryl; R¹⁷ is —OH, —F, —Cl,—Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂, —N(H)C(O)H, —N(H)C(O)NH₂,—S(O₂)NH₂, —C(O)H, -alkyl, -cycloalkyl, -perfluoroalkyl, —O-alkyl,—N(H)alkyl, —N(alkyl)₂, —CO₂alkyl, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S(O)₂N(H)alkyl,—S(O)₂N(alkyl)₂, —S-alkyl, —S(O)₂alkyl, or —C(O)alkyl; R¹⁸ is -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl, —O-heteroaryl,—O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂, —N(H)heteroaryl,—N(H)heteroaryl)₂, —N(H)alkylaryl, —N(alkylaryl)₂, —N(H)alkylheteroaryl,—N(alkylheteroaryl)2, —S-aryl, —S-heteroaryl, —S-alkylaryl,—S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkyheteroaryl, —CO₂aryl, —CO₂heteroaryl, —CO₂alkylaryl,—CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂, —C(O)N(H)heteroaryl,—C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl, —C(O)N(alkylaryl)₂,—C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂, —s(O)₂aryl,—S(O)₂heteroaryl, —S(O)₂alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂N(H)aryl, —S(O)₂N(H)heteroaryl, —S(O)₂N(H)alkylaryl,—S(O₂)N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O₂)N(heteroaryl)₂,—S(O)₂N(alkylaryl)₂, —S(O)₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroary₂, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂,—N(H)C(O)N(alkylheteroaryl)₂; R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, or -alkylheterocycle; R²⁰ is-alkyl-R¹⁸; m is 0 or 1; and X is O or S.
 3. The compound according toclaims 1 or 2, wherein: C is benzo, pyrido, thieno, pyrrolo, furo,imidazo, thiazolo, oxazolo, pyrazolo, isothiazolo, isoxazolo, ortriazolo, wherein any hydrogen bound to any ring atom is optionallyreplaced by R⁴.
 4. The compound according to claim 3, wherein: Y is

C is benzo, wherein any hydrogen bound to any ring atom is optionallyreplaced by R⁴; R¹ is -phenyl, -naphthyl, or -isoquinolinyl, wherein R¹⁷is —OH, —NH₂, —Cl, —F, -Oalkyl, or —N(alkyl)₂; R² is —C(O)—, —S(O)₂—,—C(O)C(O)—, or —CH₂C(O)—; R³ is -methyl, -ethyl, -n-propyl, -isopropyl,-phenyl, -2-pyridinyl, -3-pyridinyl, -4-pyridinyl, or -thiazolyl; R⁴ is-fluoro or -chloro; R⁵ is —OH; R⁶ is —H or —R¹⁴, wherein X═O; providedthat when —R¹⁴ is (i), R¹⁷ is -Oalkyl, —F or —Cl, and provided that when—R¹⁴ is (ii), R¹⁸ is -aryl, wherein aryl is phenyl; R⁷ is —C(O)alkyl; R⁸is -methyl, -ethyl, -n-propyl, -isopropyl, -cyclopentyl, -phenethyl, or-benzyl; X is O; m is
 0. 5. The compound according to claim 4 selectedfrom:


6. A pharmaceutical composition comprising a compound according to anyone of claims 1-5 in an amount effective for treating or preventing anIL-1 mediated disease and a pharmaceutically acceptable carrier,adjuvant or vehicle.
 7. The pharmaceutical composition according toclaim 6, wherein the IL-1-mediated disease is an inflammatory diseaseselected from the group consisting of osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, and adult respiratorydistress syndrome.
 8. The pharmaceutical composition according to claim7, wherein the inflammatory disease is osteoarthritis or acutepancreatitis.
 9. The pharmaceutical composition according to claim 6,wherein the IL-1-mediated disease is an autoimmune disease selected fromthe group consisting of glomeralonephritis, rheumatoid arthritis,systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave'sdisease, autoimmune gastritis, insulin-dependent diabetes mellitus (TypeI), autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myasthenia gravis,inflammatory bowel disease, Crohn's disease, psoriasis, and graft vshost disease.
 10. The pharmaceutical composition according to claim 9,wherein the autoimmune disease is rheumatoid arthritis, inflammatorybowel disease, or Crohn's disease, or psoriasis.
 11. The pharmaceuticalcomposition according to claim 6, wherein the IL-1-mediated disease is adestructive bone disorder selected from the group consisting ofosteoporosis or multiple myeloma-related bone disorder.
 12. Thepharmaceutical composition according to claim 6, wherein theIL-1-mediated disease is a proliferative disorder selected from thegroup consisting of acute myelogenous leukemia, chronic myelogenousleukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.13. The pharmaceutical composition according to claim 6, wherein theIL-1-mediated disease is an infectious disease, selected from the groupconsisting of sepsis, septic shock, and Shigellosis.
 14. Thepharmaceutical composition according to claim 6, wherein theIL-1-mediated disease is a degenerative or necrotic disease, selectedfrom the group consisting of Alzheimer's disease, Parkinson's disease,cerebral ischemia, and myocardial ischemia.
 15. The pharmaceuticalcomposition according to claim 14, wherein the degenerative disease isAlzheimer's disease.
 16. A pharmaceutical composition comprising acompound according to any one of claims 1-5 in an amount effective fortreating or preventing an apoptosis-mediated disease and apharmaceutically acceptable carrier, adjuvant or vehicle.
 17. Thepharmaceutical composition according to claim 16, wherein theapoptosis-mediated disease is a degenerative disease, selected from thegroup consisting of Alzheimer's disease, Parkinson's disease, cerebralischemia, myocardial ischemia, spinal muscular atrophy, multiplesclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging,alopecia, and neurological damage due to stroke.
 18. A pharmaceuticalcomposition for inhibiting an ICE-mediated function comprising acompound according to any one of claims 1-5 and a pharmaceuticallyacceptable carrier.
 19. A pharmaceutical composition for decreasing IGIFproduction comprising a compound according to any one of claims 1-5 anda pharmaceutically acceptable carrier.
 20. A pharmaceutical compositionfor decreasing IFN-γ production comprising a compound according to anyone of claims 1-5 and a pharmaceutically acceptable carrier.
 21. Apharmaceutical composition for treating or preventing a disease mediatedby excess dietary alcohol intake.
 22. A pharmaceutical composition fortreating or preventing a disease mediated by a virus.
 23. Apharmaceutical composition according to claim 22, wherein the virus isHBV, HCV, HGV, yellow fever virus, dengue fever virus, or Japaneseencephalitis virus.
 24. A method for treating or preventing a diseaseselected from an IL-1 mediated disease, an apoptosis mediated disease,an inflammatory disease, an autoimmune disease, a destructive bonedisorder, a proliferative disorder, an infectious disease, adegenerative disease, a necrotic disease, an excess dietary alcoholintake disease, a viral mediated disease, osteoarthritis, pancreatitis,asthma, adult respiratory distress syndrome, glomeralonephritis,rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Grave's disease, autoimmune gastritis, insulin-dependentdiabetes mellitus (Type I), autoimmune hemolytic anemia, autoimmuneneutropenia, thrombocytopenia, chronic active hepatitis, myastheniagravis, inflammatory bowel disease, Crohn's disease, psoriasis, graft vshost disease, osteoporosis, multiple myeloma-related bone disorder,acute myelogenous leukemia, chronic myelogenous leukemia, metastaticmelanoma, Kaposi's sarcoma, multiple myeloma, sepsis, septic shock,Shigellosis, Alzheimer's disease, Parkinson's disease, cerebralischemia, myocardial ischemia, spinal muscular atrophy, multiplesclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging,alopecia, neurological damage due to stroke, hepatitis-B, hepatitis-C,hepatitis-G, yellow fever, dengue fever, or Japanese encephalitis, in apatient comprising the step of administering to said patient apharmaceutical composition according to any one of claims 6 to
 20. 25.The method according to claim 24, wherein the disease is osteoarthritis,acute pancreatitis, rheumatoid arthritis, inflammatory bowel disease,Crohn's disease, psoriasis, or Alzeheimer's disease.
 26. A method forinhibiting an ICE-mediated function in a patient comprising the step ofadministering to said patient a pharmaceutical composition according toclaim
 18. 27. A method for decreasing IGIF or IFN-γ production in apatient comprising the step of administering to said patient apharmaceutical composition according to claim 19 or
 20. 28. A processfor preparing a compound represented by formula (V):

wherein: R²¹ is:

C is an aryl or a heteroaryl ring, wherein any hydrogen bound to anyring atom is optionally replaced by R⁴; R²² is:

each R²³ is independently -alkyl, -cycloalkyl, -aryl, -heteroaryl,-alkylaryl, -alkylheteroaryl, or -alkylheterocycle; R¹ is -aryl,-heteroaryl, -alkylaryl, or -alkylheteroaryl; R² is a bond, —C(O)—,—C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—, —N(H)S(O)₂—, —N(H)C(O)C(O)—,—CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—, —N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—,—N(R¹⁹)C(O)C(O)—, or —N(R¹⁹)CH₂C(O), or —C(O)C(═NOR¹¹)—, provided thatwhen R² is not a bond, R² is bonded to the 7-membered ring NH groupthrough carbonyl or sulfonyl; R³ is -aryl, -heteroaryl, -cycloalkyl,-alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H) (alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or CH₂N(alkyl)₂; R¹¹ is—H, -alkyl, -aryl, -heteroaryl, -cycloalkyl, -alkylaryl, or-alkylheteroaryl; R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl, -heteroaryl,-alkylaryl, -alkylheteroaryl, or -alkylheterocycle; and m is 1 or 2;comprising the steps of: a) reacting a compound represented by formula(VI): R²¹—OH, wherein R²¹ is as defined above, with a compoundrepresented by formula (VII):

wherein R²³ is as defined above, in the presence of an inert solvent,triphenylphoshine, a nucleophilic scavenger, and tetrakis-triphenylphosphine palladium(0) at ambient temperature under an inert atmosphere;and b) adding to the mixture formed in step a), HOBT and EDC.
 29. Theprocess according to claim 28, wherein: C is benzo, pyrido, thieno,pyrrolo, furo, imidazo, thiazolo, oxazolo, pyrazolo, isothiazolo,isoxazolo, or triazolo, wherein any hydrogen bound to any ring atom isoptionally replaced by R⁴.
 30. The process according to claim 29,wherein: C is benzo, wherein any hydrogen bound to any ring atom isoptionally replaced by R⁴; R¹ is phenyl, naphthyl, or isoquinolinyl,wherein R¹⁷ is —OH, —NH₂, —Cl, —F, -Oalkyl, or —N(alkyl)₂; R² is —C(O)—,—S(O)₂—, —C(O)C(O)—, or —CH₂C(O)—; R³ is methyl, ethyl, n-propyl,isopropyl, phenyl, or thiazolyl; R⁴ is -fluoro or -chloro; and m is 1.31. The process according to any one of claims, 28-30 wherein the inertsolvent is CH₂Cl₂, DMF, or a mixture of CH₂Cl₂ and DMF.
 32. The processaccording to any one of claims 28-30, wherein the nucleophilic scavengeris dimedone, morpholine, or dimethyl barbituric acid.
 33. The processaccording to claim 32, wherein the nucleophilic scavenger is dimethylbarbituric acid.
 34. The process according to claim 32, wherein theinert solvent is CH₂Cl₂, DMF, or a mixture of CH₂Cl₂ and DMF.
 35. Theprocess according to claim 34, wherein the nucleophilic scavenger isdimethyl barbituric acid.